Plant transformed with polynucleotide encoding lepidopteran-active bacillus thuringiensis delta-endotoxin

ABSTRACT

Disclosed are  Bacillus thuringiensis  strains comprising novel crystal proteins which exhibit insecticidal activity against lepidopteran insects. Also disclosed are novel  B. thuringiensis  genes and their encoded crystal proteins, as well as methods of making and using transgenic cells comprising the novel nucleic acid sequences of the invention.

This application claims the benefit of priority from U.S. ProvisionalApplication No. 60/153,995, filed Sep. 15, 1999, the entire of contentsof which is hereby specifically incorporated by reference.

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of molecularbiology. More particularly, certain embodiments concern methods andcompositions comprising DNA segments, and proteins derived frombacterial species. More particularly, it concerns novel genes fromBacillus thuringiensis encoding lepidopteran-toxic crystal proteins.Various methods for making and using these DNA segments, DNA segmentsencoding synthetically-modified Cry proteins, and native and syntheticcrystal proteins are disclosed, such as, for example, the use of DNAsegments as diagnostic probes and templates for protein production, andthe use of proteins, fusion protein carriers and peptides in variousimmunological and diagnostic applications. Also disclosed are methods ofmaking and using nucleic acid segments in the development of transgenicplant cells containing the DNA segments disclosed herein.

1.2 Description of the Related Art

Almost all field crops, plants, and commercial farming areas aresusceptible to attack by one or more insect pests. Particularlyproblematic are Coleopteran and Lepidoptern pests. For example,vegetable and cole crops such as artichokes, kohlrabi, arugula, leeks,asparagus, lentils, beans, lettuce (e.g., head, leaf, romaine), beets,bok choy, malanga, broccoli, melons (e.g., muskmelon, watermelon,crenshaw, honeydew, cantaloupe), brussels sprouts, cabbage, cardoni,carrots, napa, cauliflower, okra, onions, celery, parsley, chick peas,parsnips, chicory, peas, chinese cabbage, peppers, collards, potatoes,cucumber, pumpkins, cucurbits, radishes, dry bulb onions, rutabaga,eggplant, salsify, escarole, shallots, endive, soybean, garlic, spinach,green onions, squash, greens, sugar beets, sweet potatoes, turnip, swisschard, horseradish, tomatoes, kale, turnips, and a variety of spices aresensitive to infestation by one or more of the following insect pests:alfalfa looper, armyworm, beet armyworm, artichoke plume moth, cabbagebudworm, cabbage looper, cabbage webworm, corn earworm, celeryleafeater, cross-striped cabbageworm, european corn borer, diamondbackmoth, green cloverworm, imported cabbageworm, melonworm, omnivorousleafroller, pickleworm, rindworm complex, saltmarsh caterpillar, soybeanlooper, tobacco budworm, tomato fruitworm, tomato hornworm, tomatopinworm, velvetbean caterpillar, and yellowstriped armyworm. Likewise,pasture and hay crops such as alfalfa, pasture grasses and silage areoften attacked by such pests as armyworm, beef armyworm, alfalfacaterpillar, European skipper, a variety of loopers and webworms, aswell as yellowstriped armyworms.

Fruit and vine crops such as apples, apricots, cherries, nectarines,peaches, pears, plums, prunes, quince almonds, chestnuts, filberts,pecans, pistachios, walnuts, citrus, blackberries, blueberries,boysenberries, cranberries, currants, loganberries, raspberries,strawberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate,pineapple, tropical fruits are often susceptible to attack anddefoliation by achema sphinx moth, amorbia, armyworm, citrus cutworm,banana skipper, blackheaded fireworm, blueberry leafroller, cankerworm,cherry fruitworm, citrus cutworm, cranberry girdler, eastern tentcaterpillar, fall webworm, fall webworm, filbert leafroller, filbertwebworm, fruit tree leafroller, grape berry moth, grape leaffolder,grapeleaf skeletonizer, green fruitworm, gummosos-batrachedra commosae,gypsy moth, hickory shuckworm, hornworms, loopers, navel orangeworm,obliquebanded leafroller, omnivorous leafroller. omnivorous looper,orange tortrix, orangedog, oriental fruit moth, pandemis leafroller,peach twig borer, pecan nut casebearer, redbanded leafroller, redhumpedcaterpillar, roughskinned cutworm, saltmarsh caterpillar, spanworm, tentcaterpillar, thecla-thecla basillides, tobacco budworm, tortrix moth,tufted apple budmoth, variegated leafroller, walnut caterpillar, westerntent caterpillar, and yellowstriped armyworm.

Field crops such as canola/rape seed, evening primrose, meadow foam,corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice,safflower, small grains (barley, oats, rye, wheat, etc.), sorghum,soybeans, sunflowers, and tobacco are often targets for infestation byinsects including armyworm, asian and other corn borers, bandedsunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm(including southern and western varieties), cotton leaf perforator,diamondback moth, european corn borer, green cloverworm, headmoth,headworm, imported cabbageworm, loopers (including Anacamptodes spp.),obliquebanded leafroller, omnivorous leaftier, podworm, podworm,saltmarsh caterpillar, southwestern corn borer, soybean looper, spottedcutworm, sunflower moth, tobacco budworm, tobacco hornworm, velvetbeancaterpillar.

Bedding plants, flowers, ornamentals, vegetables and container stock arefrequently fed upon by a host of insect pests such as armyworm, azaleamoth, beet armyworm, diamondback moth, ello moth (hornworm), Floridafern caterpillar, Io moth, loopers, oleander moth, omnivorousleafroller, omnivorous looper, and tobacco budworm.

Forests, fruit, ornamental, and nut-bearing trees, as well as shrubs andother nursery stock are often susceptible to attack from diverse insectssuch as bagworm, blackheaded budworm, browntail moth, californiaoakworm, douglas fir tussock moth, elm spanworm, fall webworm, fruittreeleafroller, greenstriped mapleworm, gypsy moth, jack pine budworm,mimosa webworm, pine butterfly, redhumped caterpillar, saddlebackcaterpillar, saddle prominent caterpillar, spring and fall cankerworm,spruce budworm, tent caterpillar, tortrix, and western tussock moth.Likewise, turf grasses are often attacked by pests such as armyworm, sodwebworm, and tropical sod webworm.

Because crops of commercial interest are often the target of insectattack, environmentally-sensitive methods for controlling or eradicatinginsect infestation are desirable in many instances. This is particularlytrue for farmers, nurserymen, growers, and commercial and residentialareas which seek to control insect populations using eco-friendlycompositions.

The most widely used environmentally-sensitive insecticidal formulationsdeveloped in recent years have been composed of microbial pesticidesderived from the bacterium Bacillus thuringiensis. B. thuringiensis is aGram-positive bacterium that produces crystal proteins or inclusionbodies which are specifically toxic to certain orders and species ofinsects. Many different strains of B. thuringiensis have been shown toproduce insecticidal crystal proteins. Compositions including B.thuringiensis strains which produce insecticidal proteins have beencommercially-available and used as environmentally-acceptableinsecticides because they are quite toxic to the specific target insect,but are harmless to plants and other non-targeted organisms.

1.2.1 B. thuringiensis Crystal Proteins

1.2.1 δ-Endotoxins

δ-endotoxins are used to control a wide range of leaf-eatingcaterpillars and beetles, as well as mosquitoes. These proteinaceousparasporal crystals, also referred to as insecticidal crystal proteins,crystal proteins, Bt inclusions, crystalline inclusions, inclusionbodies, and Bt toxins, are a large collection of insecticidal proteinsproduced by B. thuringiensis that are toxic upon ingestion by asusceptible insect host. Over the past decade research on the structureand function of B. thuringiensis toxins has covered all of the majortoxin categories, and while these toxins differ in specific structureand function, general similarities in the structure and function areassumed. Based on the accumulated knowledge of B. thuringiensis toxins,a generalized mode of action for B. thuringiensis toxins has beencreated and includes: ingestion by the insect, solubilization in theinsect midgut (a combination stomach and small intestine), resistance todigestive enzymes sometimes with partial digestion actually “activating”the toxin, binding to the midgut cells, formation of a pore in theinsect cells and the disruption of cellular homeostasis (English andSlatin, 1992).

One of the unique features of B. thuringiensis is its production ofcrystal proteins during sporulation which are specifically toxic tocertain orders and species of insects. Many different strains of B.thuringiensis have been shown to produce insecticidal crystal proteins.Compositions including B. thuringiensis strains which produce proteinshaving insecticidal activity against lepidopteran and dipteran insectshave been commercially available and used as environmentally-acceptableinsecticides because they are quite toxic to the specific target insect,but are harmless to plants and other non-targeted organisms.

The mechanism of insecticidal activity of the B. thuringiensis crystalproteins has been studied extensively in the past decade. It has beenshown that the crystal proteins are toxic to the insect only afteringestion of the protein by the insect. The alkaline pH and proteolyticenzymes in the insect mid-gut solubilize the proteins, thereby allowingthe release of components which are toxic to the insect. These toxiccomponents disrupt the mid-gut cells, cause the insect to cease feeding,and, eventually, bring about insect death. For this reason, B.thuringiensis has proven to be an effective and environmentally safeinsecticide in dealing with various insect pests.

As noted by Höfte and Whiteley (1989), the majority of insecticidal B.thuringiensis strains are active against insects of the orderLepidoptera, i.e., caterpillar insects. Other B. thuringiensis strainsare insecticidally active against insects of the order Diptera, i.e.,flies and mosquitoes, or against both lepidopteran and dipteran insects.In recent years, a few B. thuringiensis strains have been reported asproducing crystal proteins that are toxic to insects of the orderColeoptera, i.e., beetles (Krieg et al., 1983; Sick et al., 1990;Donovan et al., 1992; Lambert et al., 1992a; 1992b).

1.2.2 Genes Encoding Crystal Proteins

Many of the δ-endotoxins are related to various degrees by similaritiesin their amino acid sequences. Historically, the proteins and the geneswhich encode them were classified based largely upon their spectrum ofinsecticidal activity. The review by Höfte and Whiteley (1989) discussesthe genes and proteins that were identified in B. thuringiensis prior to1990, and sets forth the nomenclature and classification scheme whichhas traditionally been applied to B. thuringiensis genes and proteins.cryI genes encode lepidopteran-toxic CryI proteins. cryII genes encodeCryII proteins that are toxic to both lepidopterans and dipterans.cryIII genes encode coleopteran-toxic CryIII proteins, while cryIV genesencode dipteran-toxic CryIV proteins. Based on the degree of sequencesimilarity, the proteins were further classified into subfamilies; morehighly related proteins within each family were assigned divisionalletters such as CryIA, CryIB, CryIC, etc. Even more closely relatedproteins within each division were given names such as CryIC1, CryIC2,etc.

Recently, a new nomenclature was developed which systematicallyclassified the Cry proteins based upon amino acid sequence homologyrather than upon insect target specificities (Crickmore et al., 1998).The classification scheme for many known toxins, including allelicvariations in individual proteins, is summarized and regularly updatedat http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/. The informationwas most recently updated as of Apr. 27, 1999 and is herein incorporatedby reference.

1.2.3 Crystal Proteins Toxic to Lepidopteran Insects 2.0 SUMMARY OF THEINVENTION

The recent review by Schnepf et al. (1998) describes the enormousdiversity of insecticidal crystal proteins derived from B.thuringiensis. Cry proteins of the Cry1, Cry2, and Cry9 classes areparticularly known for their toxicity towards lepidopteran larvae,however, the degree of toxicity varies significantly depending on thetarget lepidopteran pest (Höfte and Whiteley, 1989). For instance,Cry1Ac shows poor toxicity towards the armyworm, Spodoptera littoralis,but strong toxicity towards the tobacco budworm, Heliothis virescens. Inaddition, slight variations in amino acid sequence within a Cry proteinclass can dramatically impact insecticidal activity (see Schnepf et al.,1998 and references therein). The Cry3Ba and Cry3Bb genes, for instance,share 94% amino acid sequence identity, but only Cry3Bb exhibitssignificant toxicity towards the Southern corn rootworm, Diabroticaundecimpunctata howardi (Donovan et al., 1992). Similarly, Cry2Aa andCry2Ab share 87% amino acid sequence identity, yet only Cry2Aa displaystoxicity towards mosquitoes (Widner and Whiteley, 1990). Von Tersch etal. (1991) demonstrated that Cry1Ac proteins varying by only seven aminoacids (>99% sequence identity) nevertheless show significant differencesin insecticidal activity. Lee et al. (1996) reported that Cry1Ab allelesdiffering at only two amino acid positions exhibited a 10-folddifference in toxicity towards the gypsy moth, Lymantria dispar. Thus,even Cry proteins that are considered to be alleles of known Cryproteins or to belong to a Cry protein subclass (Crickmore et al., 1998)may have unique and useful insecticidal properties. International PatentApplication Publication No. WO 98/00546 and WO 98/40490 describe avariety of Cry1-, Cry2-, and Cry9-related crystal proteins obtained fromB. thuringiensis.

2.1 Cry DNA Segments

The present invention concerns nucleic acid segments, that can beisolated from virtually any source, that are free from total genomic DNAand that encode the novel peptides disclosed herein. Nucleic acidsegments encoding these polypeptides may encode active proteins,peptides or peptide fragments, polypeptide subunits, functional domains,or the like of one or more crystal proteins. In addition the inventionencompasses nucleic acid segments which may be synthesized entirely invitro using methods that are well-known to those of skill in the artwhich encode the novel Cry polypeptides, peptides, peptide fragments,subunits, or functional domains disclosed herein.

As used herein, the term “nucleic acid segment” refers to apolynucleotide molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a nucleic acid segment encoding anendotoxin polypeptide refers to a nucleic acid segment that comprisesone or more crystal protein-encoding sequences yet is isolated awayfrom, or purified free from, total genomic DNA of the species from whichthe nucleic acid segment is obtained, which in the instant case is thegenome of the Gram-positive bacterial genus, Bacillus, and inparticular, the species of Bacillus known as B. thuringiensis. Includedwithin the term “nucleic acid segment”, are polynucleotide segments andsmaller fragments of such segments, and also recombinant vectors,including, for example, plasmids, cosmids, phagemids, phages, viruses,and the like.

Similarly, a DNA segment comprising an isolated or purified crystalprotein-encoding gene refers to a DNA segment which may include inaddition to peptide encoding sequences, certain other elements such as,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein-encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein-,polypeptide- or peptide-encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences, operonsequences and smaller engineered gene segments that express, or may beadapted to express, proteins, polypeptides or peptides. Also, the termincludes an expression cassette comprising at least a promoter operablylinked to one or more protein coding sequences, operably linked to atleast a transcriptional termination sequence.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a nucleic acid segment or gene encodingall or part of a bacterial insecticidal crystal protein, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalnucleic acid segments or genes or operon coding regions. Of course, thisrefers to the DNA segment as originally isolated, and does not excludegenes, recombinant genes, synthetic linkers, or coding regions lateradded to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode a Crypeptide species that includes within its amino acid sequence an aminoacid sequence essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO. 63.

The term “a sequence essentially as set forth in SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:6,” for example, means that the sequencesubstantially corresponds to a portion of the sequence of SEQ ID NO:2,SEQ ID NO:4, or SEQ ID NO:6 and has relatively few amino acids that arenot identical with, or a biologically functional equivalent of, theamino acids of any of these sequences. The term “biologically functionalequivalent” is well understood in the art and is further defined indetail herein (e.g., see Illustrative Embodiments). Accordingly,sequences that have from about 70% to about 80%, or more preferablyabout 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90%, or even morepreferably about 91, 92, 93, 94, 95, 96, 97, 98, or about 99% amino acidsequence identity or functional equivalence to the amino acids of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO. 63will be sequences that are “essentially as set forth in SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO: 63.”

In addition, sequences that have from about 70% to about 80%, or morepreferably about 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90%, oreven more preferably about 91, 92, 93, 94, 95, 96, 97, 98, or about 99%nucleic acid sequence identity or functional equivalence to the nucleicacids of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49 and SEQ ID NO:62 will be sequences that are “essentially as setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:49 and SEQ ID NO:62.”

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding any of the peptide sequencesdisclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50 and SEQ ID NO: 63, or that are identical with or complementary toDNA sequences which encode any of the peptides disclosed in SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO: 63, andparticularly those DNA segments disclosed in SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ IDNO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62. For example, DNAsequences such as about 18 nucleotides, and that are up to about 10,000,about 5,000, about 3,000, about 2,000, about 1,000, about 500, about200, about 100, about 50, and about 14 base pairs in length (includingall intermediate lengths) are also contemplated to be useful.

It will be readily understood that “intermediate lengths”, in thesecontexts, means any length between the quoted ranges, such as 18, 19,20, 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101,102, 103, etc.; 150, 151, 152, 153, etc.; including all integers in theranges of from about 200-500; 500-1,000; 1,000-2,000; 2,000-3,000;3,000-5,000; and up to and including sequences of about 10,00 or sonucleotides and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequences of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50 and SEQ ID NO: 63,including those DNA sequences which are particularly disclosed in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62.Recombinant vectors and isolated DNA segments may therefore variouslyinclude the peptide-coding regions themselves, coding regions bearingselected alterations or modifications in the basic coding region, orthey may encode larger polypeptides that nevertheless include thesepeptide-coding regions or may encode biologically functional equivalentproteins or peptides that have variant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon degeneracy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.2 Cry DNA Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of crystal proteinsor peptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least a 14 nucleotidelong contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000, 2000, 5000 bp, etc. (including all intermediate lengths andup to and including the full-length gene sequences encoding eachpolypeptide will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize tocrystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of about 14 to about 17 or so, 18-25, 26-35, 36-50,or even up to and including sequences of about 100-200 nucleotides orso, identical or complementary to DNA sequences of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49 and SEQ ID NO:62, areparticularly contemplated as hybridization probes for use in, e.g.,Southern and Northern blotting. Smaller fragments will generally finduse in hybridization embodiments, wherein the length of the contiguouscomplementary region may be varied, such as between about 10-14 andabout 100 to 200 or so nucleotides, but larger contiguouscomplementarity stretches may be used, according to the lengthcomplementary sequences one wishes to detect.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating crystalprotein-encoding DNA segments. Detection of DNA segments viahybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporatedherein by reference) are exemplary of the methods of hybridizationanalyses. Teachings such as those found in the texts of Maloy et al.,1990; Maloy 1994; Segal, 1976; Prokop, 1991; and Kuby, 1991, areparticularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmentally undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.3 Vectors and Methods for Recombinant Expression of Cry Polypeptides

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or plant cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type, organism, or even animal, chosenfor expression. The use of promoter and cell type combinations forprotein expression is generally known to those of skill in the art ofmolecular biology, for example, see Sambrook et al., 1989. The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides. Appropriate promoter systemscontemplated for use in high-level expression include, but are notlimited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful. Such peptideepitopes may be amino acid sequences which comprise contiguous aminoacid sequences from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50 and SEQ ID NO: 63.

2.4 Cry Transgenes and Transgenic Plants Expressing Cry Polypeptides

In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid segmentencoding the novel polypeptides and endotoxins of the present invention.The process of producing transgenic plants is well-known in the art. Ingeneral, the method comprises transforming a suitable host cell with aDNA segment which contains a promoter operatively linked to a codingregion that encodes one or more CryET31, CryET40, CryET43, CryET44,CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54,CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63,CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83polypeptides. Such a coding region is generally operatively linked to atranscription-terminating region, whereby the promoter is capable ofdriving the transcription of the coding region in the cell, and henceproviding the cell the ability to produce the polypeptide in vivo.Alternatively, in instances where it is desirable to control, regulate,or decrease the amount of a particular recombinant crystal proteinexpressed in a particular transgenic cell, the invention also providesfor the expression of crystal protein antisense mRNA. The use ofantisense mRNA as a means of controlling or decreasing the amount of agiven protein of interest in a cell is well-known in the art.

Another aspect of the invention comprises transgenic plants whichexpress a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant” is intended to refer to a plant that has incorporatedDNA sequences, including but not limited to genes which are perhaps notnormally present, DNA sequences not normally transcribed into RNA ortranslated into a protein (“expressed”), or any other genes or DNAsequences which one desires to introduce into the non-transformed plant,such as genes which may normally be present in the non-transformed plantbut which one desires to either genetically engineer or to have alteredexpression.

It is contemplated that in some instances either the nuclear orplastidic genome, or both, of a transgenic plant of the presentinvention will have been augmented through the stable introduction ofone or more cryET31, cryET40, cryET43, cryET44, cryET45, cryET46,cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET55, cryET56,cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66,cryET67, cryET68, cryET72, cryET73, and cryET83 transgenes, eithernative, synthetically modified, or mutated. In some instances, more thanone transgene will be incorporated into one or more genomes of thetransformed host plant cell. Such is the case when more than one crystalprotein-encoding DNA segment is incorporated into the genome of such aplant. In certain situations, it may be desirable to have one, two,three, four, or even more B. thuringiensis crystal proteins (eithernative or recombinantly-engineered) incorporated and stably expressed inthe transformed transgenic plant.

A preferred gene which may be introduced includes, for example, acrystal protein-encoding DNA sequence from bacterial origin, andparticularly one or more of those described herein which are obtainedfrom Bacillus spp. Highly preferred nucleic acid sequences are thoseobtained from B. thuringiensis, or any of those sequences which havebeen genetically engineered to decrease or increase the insecticidalactivity of the crystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of a transgeniccell line are well-known in the art, and are discussed herein. Vectors,plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segmentsfor use in transforming such cells will, of course, generally compriseeither the operons, genes, or gene-derived sequences of the presentinvention, either native, or synthetically-derived, and particularlythose encoding the disclosed crystal proteins. These DNA constructs canfurther include structures such as promoters, enhancers, polylinkers, oreven gene sequences which have positively- or negatively-regulatingactivity upon the particular genes of interest as desired. The DNAsegment or gene may encode either a native or modified crystal protein,which will be expressed in the resultant recombinant cells, and/or whichwill impart an improved phenotype to the regenerated plant.

Such transgenic plants may be desirable for increasing the insecticidalresistance of a monocotyledonous or dicotyledonous plant, byincorporating into such a plant, a transgenic DNA segment encoding oneor more CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47,CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56, CryET57,CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67,CryET68, CryET72, CryET73, and CryET83 polypeptides which are toxic to alepidopteran insect. Particularly preferred plants include turf grasses,kapok, sorghum, cotton, corn, soybeans, oats, rye, wheat, flax, tobacco,rice, tomatoes, potatoes, or other vegetables, ornamental plants, fruittrees, and the like.

In a related aspect, the present invention also encompasses a seedproduced by the transformed plant, a progeny from such seed, and a seedproduced by the progeny of the original transgenic plant, produced inaccordance with the above process. Such progeny and seeds will have acrystal protein-encoding transgene stably incorporated into theirgenome, and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more CryET31, CryET40, CryET43, CryET44,CryET45, CryET46, CryET47, CryET49, CryET51, CryET52, CryET53, CryET54,CryET55, CryET56, CryET57, CryET59, CryET60, CryET61, CryET62, CryET63,CryET64, CryET66, CryET67, CryET68, CryET72, CryET73, and CryET83crystal proteins or polypeptides are aspects of this invention. Aswell-known to those of skill in the art, a progeny of a plant isunderstood to mean any offspring or any descendant from such a plant,but in this case means any offspring or any descendant which alsocontains the transgene.

2.5 Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. The technique of site-specific mutagenesis is well knownin the art, as exemplified by various publications.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the endotoxin-encoding nucleicacid segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

2.6 Antibody Compositions and Methods of Making

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal (mAbs) or polyclonal which bind to one ormore of the polypeptides disclosed herein. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g., Harlowand Lane, 1988; incorporated herein by reference). mAbs may be readilyprepared through use of well-known techniques, such as those exemplifiedin U.S. Pat. No. 4,196,265, incorporated herein by reference.

2.7 ELISAs and Immunoprecipitation

ELISAs may be used in conjunction with the invention. Many differentprotocols exist for performing ELISAs. These are well known to those ofordinary skill in the art. Examples of basic ELISA protocols may befound in any standard molecular biology laboratory manual (e.g.Sambrook, Fritsch, and Maniatis, eds. Molecular cloning: a laboratorymanual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 1989).

2.8 Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. Methods of performing immunoblotand western blot analysis are well known to those of skill in the are(see Sambrook, et al, ibid). Immunologically-based detection methods foruse in conjunction with Western blotting include enzymatically-,radiolabel-, or fluorescently-tagged secondary antibodies against thetoxin moiety are considered to be of particular use in this regard.

2.9 Crystal Protein Screening and Detection Kits

The present invention contemplates methods and kits for screeningsamples suspected of containing crystal protein polypeptides or crystalprotein-related polypeptides, or cells producing such polypeptides. Akit may contain one or more antibodies of the present invention, and mayalso contain reagent(s) for detecting an interaction between a sampleand an antibody of the present invention. The provided reagent(s) can beradio-, fluorescently- or enzymatically-labeled or even epitope orligand tagged. The kit can contain a known radiolabeled agent capable ofbinding or interacting with a nucleic acid or antibody of the presentinvention.

The reagent(s) of the kit can be provided as a liquid solution, attachedto a solid support or as a dried powder. Preferably, when the reagent(s)are provided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent(s) provided are attached to asolid support, the solid support can be chromatograph media, a testplate having a plurality of wells, or a microscope slide. When thereagent(s) provided are a dry powder, the powder can be reconstituted bythe addition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. One may find additionaladvantages through the use of a secondary binding ligand such as asecond antibody or a biotin/avidin ligand binding arrangement, as isknown in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the tittering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides.

Generally speaking, kits in accordance with the present invention willinclude a suitable crystal protein, peptide or an antibody directedagainst such a protein or peptide, together with an immunodetectionreagent and a means for containing the antibody or antigen and reagent.The immunodetection reagent will typically comprise a label associatedwith the antibody or antigen, or associated with a secondary bindingligand. Exemplary ligands might include a secondary antibody directedagainst the first antibody or antigen or a biotin or avidin (orstreptavidin) ligand having an associated label. Of course, as notedabove, a number of exemplary labels are known in the art and all suchlabels may be employed in connection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquoted. The kits of the present invention will also typically includea means for containing the antibody, antigen, and reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

2.10 Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies. In particular, the invention concerns epitopic coresequences derived from Cry proteins or peptides.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a crystal protein or polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the crystal protein orpolypeptide will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art. The identification of Cryimmunodominant epitopes, and/or their functional equivalents, suitablefor use in vaccines is a relatively straightforward matter (e.g. U.S.Pat. No. 4,554,101; Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat.No. 4,554,101). The amino acid sequence of these “epitopic coresequences” may then be readily incorporated into peptides, eitherthrough the application of peptide synthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 8 to about 20 amino acids in length,and more preferably about 8 to about 15 amino acids in length. It isproposed that particular advantages of the present invention may berealized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to crystal proteins,and in particular CryET31, CryET40, CryET43, CryET44, CryET45, CryET46,CryET47, CryET49, CryET51, CryET52, CryET53, CryET54, CryET55, CryET56,CryET57, CryET59, CryET60, CryET61, CryET62, CryET63, CryET64, CryET66,CryET67, CryET68, CryET72, CryET73, CryET83 and related sequences. Theseepitopic core sequences are identified herein in particular aspects ashydrophilic regions of the particular polypeptide antigen.

Computerized peptide sequence analysis programs (e.g., DNAStar®software, DNAStar, Inc., Madison, Wis.) may also be useful in designingsynthetic peptides in accordance with the present disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer).

2.11 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein, andconsequently increasing the insecticidal activity and/or expression ofthe recombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 1.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

2.12 Insecticidal Compositions and Methods of Use

The inventors contemplate that the crystal protein compositionsdisclosed herein will find particular utility as insecticides fortopical and/or systemic application to field crops, grasses, fruits andvegetables, and ornamental plants. In a preferred embodiment, thebioinsecticide composition comprises an oil flowable suspension ofbacterial cells which expresses a novel crystal protein disclosedherein. Preferably the cells are B. thuringiensis NRRL B-21921, NRRLB-21922, NRRL B-21923, NRRL B-21924, NRRL B-21925, NRRL B-21926, NRRLB-21927, NRRL B-21928, NRRL B-21929, NRRL B-21930, NRRL B-21931, NRRLB-21932, NRRL B-21933, NRRL B-21934, NRRL B-21935, NRRL B-21936, NRRLB-21937, NRRL B-21938, NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRLB-21942, NRRL B-21943, and NRRL B-21944, however, any such bacterialhost cell expressing the novel nucleic acid segments disclosed hereinand producing a crystal protein is contemplated to be useful, such as B.thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.

In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel crystal protein disclosed herein.Preferred bacterial cells are B. thuringiensis NRRL B-21921, NRRLB-21922, NRRL B-21923, NRRL B-21924, NRRL B-21925, NRRL B-21926, NRRLB-21927, NRRL B-21928, NRRL B-21929, NRRL B-21930, NRRL B-21931, NRRLB-21932, NRRL B-21933, NRRL B-21934, NRRL B-21935, NRRL B-21936, NRRLB-21937, NRRL B-21938, NRRL B-21939, NRRL B-21940, NRRL B-21941, NRRLB-21942, NRRL B-21943, and NRRL B-21944, however, bacteria such as B.thuringiensis, B. megaterium, B. subtilis, E. coli, or Pseudomonas spp.cells transformed with a DNA segment disclosed herein and expressing thecrystal protein are also contemplated to be useful.

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, pellet, or colloidal concentrate.This powder comprises bacterial cells which expresses a novel crystalprotein disclosed herein. Preferred bacterial cells are B. thuringiensisNRRL B-21921, NRRL B-21922, NRRL B-21923, NRRL B-21924, NRRL B-21925,NRRL B-21926, NRRL B-21927, NRRL B-21928, NRRL B-21929, NRRL B-21930,NRRL B-21931, NRRL B-21932, NRRL B-21933, NRRL B-21934, NRRL B-21935,NRRL B-21936, NRRL B-21937, NRRL B-21938, NRRL B-21939, NRRL B-21940,NRRL B-21941, NRRL B-21942, NRRL B-21943, and NRRL B-21944 cells,however, bacteria such as B. thuringiensis, B. megaterium, B. subtilis,E. coli, or Pseudomonas spp. cells transformed with a DNA segmentdisclosed herein and expressing the crystal protein are alsocontemplated to be useful. Such dry forms of the insecticidalcompositions may be formulated to dissolve immediately upon wetting, oralternatively, dissolve in a controlled-release, sustained-release, orother time-dependent manner.

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above which express the crystal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solutionready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise intact B. thuringiensiscells expressing the protein of interest, such bacteria may beformulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various diluents, inertmaterials, such as inorganic minerals (phyllosilicates, carbonates,sulfates, phosphates, and the like) or botanical materials (powderedcorncobs, rice hulls, walnut shells, and the like). The formulations mayinclude spreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel insecticidal polypeptides may be prepared bynative or recombinant bacterial expression systems in vitro and isolatedfor subsequent field application. Such protein may be either in crudecell lysates, suspensions, colloids, etc., or alternatively may bepurified, refined, buffered, and/or further processed, beforeformulating in an active biocidal formulation. Likewise, under certaincircumstances, it may be desirable to isolate crystals and/or sporesfrom bacterial cultures expressing the crystal protein and applysolutions, suspensions, or colloidal preparations of such crystalsand/or spores as the active bioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) is applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,E. coli, inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in insecticide formulationtechnology; these are well known to those skilled in insecticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, E. coli, byhomogeneously mixing, blending and/or grinding the insecticidalcomposition with suitable adjuvants using conventional formulationtechniques.

The insecticidal compositions of this invention are applied to theenvironment of the target lepidopteran insect, typically onto thefoliage of the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, including dusting, sprinkling, soaking,soil injection, seed coating, seedling coating, spraying, aerating,misting, atomizing, and the like, are also feasible and may be requiredunder certain circumstances such as e.g., insects that cause root orstalk infestation, or for application to delicate vegetation orornamental plants. These application procedures are also well-known tothose of skill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe polypeptide compositions may be from about 1% to about 99% or moreby weight of the protein composition, while liquid formulations maygenerally comprise from about 1% to about 99% or more of the activeingredient by weight. Formulations which comprise intact bacterial cellswill generally contain from about 110 to about 110 cells/mg.

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 50g to about 500 g of active ingredient, or of from about 500 g to about1000 g, or of from about 1000 g to about 5000 g or more of activeingredient.

5.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

5.1 Some Advantages of the Invention

The use of B. thuringiensis insecticidal crystal protein genes for inplanta production of insecticidal proteins, thereby conferring insectresistance on important agronomic plants, is rapidly gaining commercialacceptance in the United States and abroad. The need for newinsecticidal traits does not diminish, however, with the successfuldeployment of a handful of cry genes in plants. Concerns over thepotential for insect resistance development, for instance, makes itimperative that an arsenal of insecticidal proteins (i.e. cry genes) beassembled to provide the genetic material necessary for tomorrow'sinsecticidal traits. In addition, transgenic plants producing a B.thuringiensis Cry protein may still be susceptible to damage fromsecondary insect pests, thus prompting the search for additional Cryproteins with improved efficacy towards these pests. The B.thuringiensis crystal proteins of the present invention represent adiverse collection of insecticidal proteins, including several that aretoxic towards a lepidopteran colony exhibiting resistance to certaintypes of Cry1 proteins. Bioassays against a wide range of lepidopteranpests confirm that these proteins possess insecticidal activity and,furthermore, that these proteins vary in efficacy against this array oftarget insects. This variation in the spectrum of insects affected bythe toxin proteins suggests differing modes of action that may beimportant for future insect resistance management strategies. In plantaexpression of the cry genes of the present invention can confer insectresistance to the host plant as has been demonstrated for other crygenes from B. thuringiensis.

5.2 Probes and Primers

In another aspect, DNA sequence information provided by the inventionallows for the preparation of relatively short DNA (or RNA) sequenceshaving the ability to specifically hybridize to gene sequences of theselected polynucleotides disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of a selected crystal protein gene sequence, e.g., asequence such as that shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49 and SEQ ID NO:62. The ability of such DNAs andnucleic acid probes to specifically hybridize to a crystalprotein-encoding gene sequence lends them particular utility in avariety of embodiments. Most importantly, the probes may be used in avariety of assays for detecting the presence of complementary sequencesin a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingPCR™ technology. Segments of related crystal protein genes from otherspecies may also be amplified by PCR™ using such primers.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes sequences that are complementary to at leasta 14 to 30 or so long nucleotide stretch of a crystal protein-encodingsequence, such as that shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49 and SEQ ID NO:62. A size of at least about 14 or sonucleotides in length helps to ensure that the fragment will be ofsufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than about 14 or so bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of about 14 to about 20 or sonucleotides, or even longer where desired. Such fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, by application of nucleic acid reproduction technology, such asthe PCR™ technology of U.S. Pat. Nos. 4,683,195, and 4,683,202, hereinincorporated by reference, or by excising selected DNA fragments fromrecombinant plasmids containing appropriate inserts and suitablerestriction sites.

5.3 Expression Vectors

The present invention contemplates an expression vector comprising apolynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to an coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region.

As used herein, the term “operatively linked” means that a promoter isconnected to an coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region are well known inthe art.

In a preferred embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is preferable in aBacillus host cell. Preferred host cells include B. thuringiensis, B.megaterium, B. subtilis, and related bacilli, with B. thuringiensis hostcells being highly preferred. Promoters that function in bacteria arewell-known in the art. An exemplary and preferred promoter for theBacillus crystal proteins include any of the known crystal protein genepromoters, including the cryET31, cryET40, cryET43, cryET44, cryET45,cryET46, cryET47, cryET49, cryET51, cryET5Z cryET53, cryET54, cryET55,cryET56, cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64,cryET66, cryET67, cryET68, cryET7Z cryET73, and cryET83 gene promoters.Alternatively, mutagenized or recombinant crystal protein-encoding genepromoters may be engineered by the hand of man and used to promoteexpression of the novel gene segments disclosed herein.

In an alternate embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is performed using atransformed Gram-negative bacterium such as an E. coli or Pseudomonasspp. host cell. Promoters which function in high-level expression oftarget polypeptides in E. coli and other Gram-negative host cells arealso well-known in the art.

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in plants. Promoters that function in plants are also wellknown in the art. Useful in expressing the polypeptide in plants arepromoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

A promoter is also selected for its ability to direct the transformedplant cell's or transgenic plant's transcriptional activity to thecoding region. Structural genes can be driven by a variety of promotersin plant tissues. Promoters can be near-constitutive, such as the CaMV35S promoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S,increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callus, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which isspecific for seed tissue. The Lectin protein in soybean seeds is encodedby a single gene (Le1) that is only expressed during seed maturation andaccounts for about 2 to about 5% of total seed mRNA. The lectin gene andseed-specific promoter have been fully characterized and used to directseed specific expression in transgenic tobacco plants (Vodkin et al.,1983; Lindstrom et al., 1990.)

An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yanget al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), cornlight harvesting complex (Simpson, 1986), corn heat shock protein (Odellet al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986;Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), CaMV 35S transcript (Odell et al., 1985) andPotato patatin (Wenzler et al., 1989). Preferred promoters include acauliflower mosaic virus (CaMV 35S) promoter, a S-E9 small subunit RuBPcarboxylase promoter, a rice actin promoter, a maize histone promoter, afused CAMV 35S-Arabidopsis histone promoter, a CaMV 35S promoter, a CaMV19S promoter, a nos promoter, an Adh promoter, an actin promoter, ahistone promoter, a ribulose bisphosphate carboxylase promoter, anR-allele promoter, a root cell promoter, an α-tubulin promoter, anABA-inducible promoter, a turgor-inducible promoter, a rbcS promoter, acorn sucrose synthetase 1 promoter, a corn alcohol dehydrogenase 1promoter, a corn light harvesting complex promoter, a corn heat shockprotein promoter, a pea small subunit RuBP carboxylase promoter, a Tiplasmid mannopine synthase promoter, a Ti plasmid nopaline synthasepromoter, a petunia chalcone isomerase promoter, a bean glycine richprotein 1 promoter, a CaMV 35S transcript promoter, a potato patatinpromoter, a cab promoter, a PEP-Carboxylase promoter and an S-E9 smallsubunit RuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al.,1987). However, several other plant integrating vector systems are knownto function in plants including pCaMVCN transfer control vectordescribed (Fromm et al., 1985). Plasmid pCaMVCN (available fromPharmacia, Piscataway, N.J.) includes the cauliflower mosaic virus CaMV35S promoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011, the disclosures of which areincorporated herein by reference. Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

A variety of methods has been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to conferinsecticidal activity to a cell is preferably a CryET31, CryET40,CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51, CryET52,CryET53, CryET54, CryET55, CryET56, CryET57, CryET59, CryET60, CryET61,CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73,and CryET83 polypeptide-encoding gene.

5.7 Nomenclature of the Novel Polypeptides

The inventors have arbitrarily assigned the designation CryET31,CryET40, CryET43, CryET44, CryET45, CryET46, CryET47, CryET49, CryET51,CryET52, CryET53, CryET54, CryET56, CryET57, CryET59, CryET60, CryET61,CryET62, CryET63, CryET64, CryET66, CryET67, CryET68, CryET72, CryET73,and CryET83 to the polypeptides of this invention. Likewise, thearbitrary designations of cryET31, cryET40, cryET43, cryET44, cryET45,cryET46, cryET47, cryET49, cryET5, cryET52, cryET53, cryET54, cryET56,cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66,cryET67, cryET68, cryET72, cryET73, and cryET83 have been assigned tothe novel nucleic acid sequence which encodes these polypeptides,respectively. Formal assignment of gene and protein designations basedon the revised nomenclature of crystal protein endotoxins will beassigned by a committee on the nomenclature of B. thuringiensis, formedto systematically classify B. thuringiensis crystal proteins. Theinventors contemplate that the arbitrarily assigned designations of thepresent invention will be superceded by the official nomenclatureassigned to these sequences.

5.8 Transformed Host Cells and Transgenic Plants

Methods and compositions for transforming a bacterium, a yeast cell, aplant cell, or an entire plant with one or more expression vectorscomprising a crystal protein-encoding gene segment are further aspectsof this disclosure. A transgenic bacterium, yeast cell, plant cell orplant derived from such a transformation process or the progeny andseeds from such a transgenic plant are also further embodiments of theinvention.

Means for transforming bacteria and yeast cells are well known in theart. Typically, means of transformation are similar to those well knownmeans used to transform other bacteria or yeast such as E. coli orSaccharomyces cerevisiae. Methods for DNA transformation of plant cellsinclude Agrobacterium-mediated plant transformation, protoplasttransformation, gene transfer into pollen, injection into reproductiveorgans, injection into immature embryos and particle bombardment. Eachof these methods has distinct advantages and disadvantages. Thus, oneparticular method of introducing genes into a particular plant strainmay not necessarily be the most effective for another plant strain, butit is well known which methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973;Zatloukal et al., 1992); (2) physical methods such as microinjection(Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al.,1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang,1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al.,1993; Eglitis and Anderson, 1988a; 1988b); and (4) receptor-mediatedmechanisms (Curiel et al., 1991; 1992; Wagner et al., 1992).

5.8.3 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNAis a relatively precise process resulting in few rearrangements. Theregion of DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., 1986; Jorgensen et al., 1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Therefore, commercially important cereal grains such asrice, corn, and wheat must usually be transformed using alternativemethods. However, as mentioned above, the transformation of asparagususing Agrobacterium can also be achieved (see, for example, Bytebier etal., 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

More preferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for enhanced carboxylase activity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada etal., 1986; Abdullah et al., 1986).

5.8.4 Other Transformation Methods

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988).

Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada etal., 1986; Abdullah et al., 1986).

5.8.5 Gene Expression in Plants

Although great progress has been made in recent years with respect topreparation of transgenic plants which express bacterial proteins suchas B. thuringiensis crystal proteins, the results of expressing nativebacterial genes in plants are often disappointing. In recent years,however, several potential factors have been implicated as responsiblein varying degrees for the level of protein expression from a particularcoding sequence. For example, scientists now know that maintaining asignificant level of a particular mRNA in the cell is indeed a criticalfactor. Unfortunately, the causes for low steady state levels of mRNAencoding foreign proteins are many. First, full length RNA synthesis maynot occur at a high frequency. This could, for example, be caused by thepremature termination of RNA during transcription or due to unexpectedmRNA processing during transcription. Second, full length RNA may beproduced in the plant cell, but then processed (splicing, polyAaddition) in the nucleus in a fashion that creates a nonfunctional mRNA.If the RNA is not properly synthesized, terminated and polyadenylated,it cannot move to the cytoplasm for translation. Similarly, in thecytoplasm, if mRNAs have reduced half lives (which are determined bytheir primary or secondary sequence) insufficient protein product willbe produced. In addition, there is an effect, whose magnitude isuncertain, of translational efficiency on mRNA half-life. In addition,every RNA molecule folds into a particular structure, or perhaps familyof structures, which is determined by its sequence. The particularstructure of any RNA might lead to greater or lesser stability in thecytoplasm. Structure per se is probably also a determinant of mRNAprocessing in the nucleus. It is likely that dramatically changing thesequence of an RNA will have a large effect on its folded structure Itis likely that structure per se or particular structural features alsohave a role in determining RNA stability.

To overcome these limitations in foreign gene expression, researchershave identified particular sequences and signals in RNAs that have thepotential for having a specific effect on RNA stability. In certainembodiments of the invention, therefore, there is a desire to optimizeexpression of the disclosed nucleic acid segments in planta. Oneparticular method of doing so, is by alteration of the bacterial gene toremove sequences or motifs which decrease expression in a transformedplant cell. The process of engineering a coding sequence for optimalexpression in planta is often referred to as “plantizing” a DNAsequence.

Particularly problematic sequences are those which are A+T rich.Unfortunately, since B. thuringiensis has an A+T rich genome, nativecrystal protein gene sequences must often be modified for optimalexpression in a plant. The sequence motif ATTTA (or AUUUA as it appearsin RNA) has been implicated as a destabilizing sequence in mammaliancell mRNA (Shaw and Kamen, 1986). Many short lived mRNAs have A+T rich3′ untranslated regions, and these regions often have the ATTTAsequence, sometimes present in multiple copies or as multimers (e.g.,ATTTATTTA . . . ). Shaw and Kamen showed that the transfer of the 3′ endof an unstable mRNA to a stable RNA (globin or VA1) decreased the stableRNA's half life dramatically. They further showed that a pentamer ofATTTA had a profound destabilizing effect on a stable message, and thatthis signal could exert its effect whether it was located at the 3′ endor within the coding sequence. However, the number of ATTTA sequencesand/or the sequence context in which they occur also appear to beimportant in determining whether they function as destabilizingsequences. Shaw and Kamen showed that a trimer of ATTTA had much lesseffect than a pentamer on mRNA stability and a dimer or a monomer had noeffect on stability (Shaw and Kamen, 1987). Note that multimers of ATTTAsuch as a pentamer automatically create an A+T rich region. This wasshown to be a cytoplasmic effect, not nuclear. In other unstable mRNAs,the ATTTA sequence may be present in only a single copy, but it is oftencontained in an A+T rich region. From the animal cell data collected todate, it appears that ATTTA at least in some contexts is important instability, but it is not yet possible to predict which occurrences ofATTTA are destabiling elements or whether any of these effects arelikely to be seen in plants.

Some studies on mRNA degradation in animal cells also indicate that RNAdegradation may begin in some cases with nucleolytic attack in A+T richregions. It is not clear if these cleavages occur at ATTTA sequences.There are also examples of mRNAs that have differential stabilitydepending on the cell type in which they are expressed or on the stagewithin the cell cycle at which they are expressed. For example, histonemRNAs are stable during DNA synthesis but unstable if DNA synthesis isdisrupted. The 3′ end of some histone mRNAs seems to be responsible forthis effect (Pandey and Marzluff, 1987). It does not appear to bemediated by ATTTA, nor is it clear what controls the differentialstability of this mRNA. Another example is the differential stability ofIgG mRNA in B lymphocytes during B cell maturation (Genovese andMilcarek, 1988). A final example is the instability of a mutantβ-thallesemic globin mRNA. In bone marrow cells, where this gene isnormally expressed, the mutant mRNA is unstable, while the wild-typemRNA is stable. When the mutant gene is expressed in HeLa or L cells invitro, the mutant mRNA shows no instability (Lim et al., 1988). Theseexamples all provide evidence that mRNA stability can be mediated bycell type or cell cycle specific factors. Furthermore this type ofinstability is not yet associated with specific sequences. Given theseuncertainties, it is not possible to predict which RNAs are likely to beunstable in a given cell. In addition, even the ATTTA motif may actdifferentially depending on the nature of the cell in which the RNA ispresent. Shaw and Kamen (1987) have reported that activation of proteinkinase C can block degradation mediated by ATTTA.

The addition of a polyadenylate string to the 3′ end is common to mosteukaryotic mRNAs, both plant and animal. The currently accepted view ofpolyA addition is that the nascent transcript extends beyond the mature3′ terminus. Contained within this transcript are signals forpolyadenylation and proper 3′ end formation. This processing at the 3′end involves cleavage of the mRNA and addition of polyA to the mature 3′end. By searching for consensus sequences near the polyA tract in bothplant and animal mRNAs, it has been possible to identify consensussequences that apparently are involved in polyA addition and 3′ endcleavage. The same consensus sequences seem to be important to both ofthese processes. These signals are typically a variation on the sequenceAATAAA. In animal cells, some variants of this sequence that arefunctional have been identified; in plant cells there seems to be anextended range of functional sequences (Wickens and Stephenson, 1984;Dean et al., 1986). Because all of these consensus sequences arevariations on AATAAA, they all are A+T rich sequences. This sequence istypically found 15 to 20 bp before the polyA tract in a mature mRNA.Studies in animal cells indicate that this sequence is involved in bothpolyA addition and 3′ maturation. Site directed mutations in thissequence can disrupt these functions (Conway and Wickens, 1988; Wickenset al., 1987). However, it has also been observed that sequences up to50 to 100 bp 3′ to the putative polyA signal are also required; i.e., agene that has a normal AATAAA but has been replaced or disrupteddownstream does not get properly polyadenylated (Gil and Proudfoot,1984; Sadofsky and Alwine, 1984; McDevitt et al., 1984). That is, thepolyA signal itself is not sufficient for complete and properprocessing. It is not yet known what specific downstream sequences arerequired in addition to the polyA signal, or if there is a specificsequence that has this function. Therefore, sequence analysis can onlyidentify potential polyA signals.

In naturally occurring mRNAs that are normally polyadenylated, it hasbeen observed that disruption of this process, either by altering thepolyA signal or other sequences in the mRNA, profound effects can beobtained in the level of functional mRNA. This has been observed inseveral naturally occurring mRNAs, with results that are gene-specificso far.

It has been shown that in natural mRNAs proper polyadenylation isimportant in mRNA accumulation, and that disruption of this process caneffect mRNA levels significantly. However, insufficient knowledge existsto predict the effect of changes in a normal gene. In a heterologousgene, it is even harder to predict the consequences. However, it ispossible that the putative sites identified are dysfunctional. That is,these sites may not act as proper polyA sites, but instead function asaberrant sites that give rise to unstable mRNAs.

In animal cell systems, AATAAA is by far the most common signalidentified in mRNAs upstream of the polyA, but at least four variantshave also been found (Wickens and Stephenson, 1984). In plants, notnearly so much analysis has been done, but it is clear that multiplesequences similar to AATAAA can be used. The plant sites in Table 2called major or minor refer only to the study of Dean et al. (1986)which analyzed only three types of plant gene. The designation ofpolyadenylation sites as major or minor refers only to the frequency oftheir occurrence as functional sites in naturally occurring genes thathave been analyzed. In the case of plants this is a very limiteddatabase. It is hard to predict with any certainty that a sitedesignated major or minor is more or less likely to function partiallyor completely when found in a heterologous gene such as those encodingthe crystal proteins of the present invention.

TABLE 2 POLYADENYLATION SITES IN PLANT GENES PA AATAAA Major consensussite P1A AATAAT Major plant site P2A AACCAA Minor plant site P3A ATATAA″ P4A AATCAA ″ P5A ATACTA ″ P6A ATAAAA ″ P7A ATGAAA ″ P8A AAGCAT ″ P9AATTAAT ″ P10A ATACAT ″ P11A AAAATA ″ P12A ATTAAA Minor animal site P13AAATTAA ″ P14A AATACA ″ P15A CATAAA ″

The present invention provides a method for preparing synthetic plantgenes which genes express their protein product at levels significantlyhigher than the wild-type genes which were commonly employed in planttransformation heretofore. In another aspect, the present invention alsoprovides novel synthetic plant genes which encode non-plant proteins.

As described above, the expression of native B. thuringiensis genes inplants is often problematic. The nature of the coding sequences of B.thuringiensis genes distinguishes them from plant genes as well as manyother heterologous genes expressed in plants. In particular, B.thuringiensis genes are very rich (˜62%) in adenine (A) and thymine (T)while plant genes and most other bacterial genes which have beenexpressed in plants are on the order of 45-55% A+T.

Due to the degeneracy of the genetic code and the limited number ofcodon choices for any amino acid, most of the “excess” A+T of thestructural coding sequences of some Bacillus species are found in thethird position of the codons. That is, genes of some Bacillus specieshave A or T as the third nucleotide in many codons. Thus A+T content inpart can determine codon usage bias. In addition, it is clear that genesevolve for maximum function in the organism in which they evolve. Thismeans that particular nucleotide sequences found in a gene from oneorganism, where they may play no role except to code for a particularstretch of amino acids, have the potential to be recognized as genecontrol elements in another organism (such as transcriptional promotersor terminators, polyA addition sites, intron splice sites, or specificmRNA degradation signals). It is perhaps surprising that such misreadsignals are not a more common feature of heterologous gene expression,but this can be explained in part by the relatively homogeneous A+Tcontent (˜50%) of many organisms. This A+T content plus the nature ofthe genetic code put clear constraints on the likelihood of occurrenceof any particular oligonucleotide sequence. Thus, a gene from E. coliwith a 50% A+T content is much less likely to contain any particular A+Trich segment than a gene from B. thuringiensis.

Typically, to obtain high-level expression of the S-endotoxin genes inplants, existing structural coding sequence (“structural gene”) whichcodes for the S-endotoxin are modified by removal of ATTTA sequences andputative polyadenylation signals by site directed mutagenesis of the DNAcomprising the structural gene. It is most preferred that substantiallyall the polyadenylation signals and ATTTA sequences are removed althoughenhanced expression levels are observed with only partial removal ofeither of the above identified sequences. Alternately if a syntheticgene is prepared which codes for the expression of the subject protein,codons are selected to avoid the ATTTA sequence and putativepolyadenylation signals. For purposes of the present invention putativepolyadenylation signals include, but are not necessarily limited to,AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT,ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA. In replacingthe ATTTA sequences and polyadenylation signals, codons are preferablyutilized which avoid the codons which are rarely found in plant genomes.

The selected DNA sequence is scanned to identify regions with greaterthan four consecutive adenine (A) or thymine (T) nucleotides. The A+Tregions are scanned for potential plant polyadenylation signals.Although the absence of five or more consecutive A or T nucleotideseliminates most plant polyadenylation signals, if there are more thanone of the minor polyadenylation signals identified within tennucleotides of each other, then the nucleotide sequence of this regionis preferably altered to remove these signals while maintaining theoriginal encoded amino acid sequence.

The second step is to consider the about 15 to about 30 or so nucleotideresidues surrounding the A+T rich region identified in step one. If theA+T content of the surrounding region is less than 80%, the regionshould be examined for polyadenylation signals. Alteration of the regionbased on polyadenylation signals is dependent upon (1) the number ofpolyadenylation signals present and (2) presence of a major plantpolyadenylation signal.

The extended region is examined for the presence of plantpolyadenylation signals. The polyadenylation signals are removed bysite-directed mutagenesis of the DNA sequence. The extended region isalso examined for multiple copies of the ATTTA sequence which are alsoremoved by mutagenesis.

It is also preferred that regions comprising many consecutive A+T basesor G+C bases are disrupted since these regions are predicted to have ahigher likelihood to form hairpin structure due to self-complementarity.Therefore, insertion of heterogeneous base pairs would reduce thelikelihood of self-complementary secondary structure formation which areknown to inhibit transcription and/or translation in some organisms. Inmost cases, the adverse effects may be minimized by using sequenceswhich do not contain more than five consecutive A+T or G+C.

5.8.6 Synthetic Oligonucleotides for Mutagenesis

When oligonucleotides are used in the mutagenesis, it is desirable tomaintain the proper amino acid sequence and reading frame, withoutintroducing common restriction sites such as BglII, HindIII, SacI, KpnI,EcoRI, NcoI, PstI and SalI into the modified gene. These restrictionsites are found in poly-linker insertion sites of many cloning vectors.Of course, the introduction of new polyadenylation signals, ATTTAsequences or consecutive stretches of more than five A+T or G+C, shouldalso be avoided. The preferred size for the oligonucleotides is about 40to about 50 bases, but fragments ranging from about 18 to about 100bases have been utilized. In most cases, a minimum of about 5 to about 8base pairs of homology to the template DNA on both ends of thesynthesized fragment are maintained to insure proper hybridization ofthe primer to the template. The oligonucleotides should avoid sequenceslonger than five base pairs A+T or G+C. Codons used in the replacementof wild-type codons should preferably avoid the TA or CG doubletwherever possible. Codons are selected from a plant preferred codontable (such as Table 3 below) so as to avoid codons which are rarelyfound in plant genomes, and efforts should be made to select codons topreferably adjust the G+C content to about 50%.

Regions with many consecutive A+T bases or G+C bases are predicted tohave a higher likelihood to form hairpin structures due toself-complementarity. Disruption of these regions by the insertion ofheterogeneous base pairs is preferred and should reduce the likelihoodof the formation of self-complementary secondary structures such ashairpins which are known in some organisms to inhibit transcription(transcriptional terminators) and translation (attenuators).

Alternatively, a completely synthetic gene for a given amino acidsequence can be prepared, with regions of five or more consecutive A+Tor G+C nucleotides being avoided. Codons are selected avoiding the TAand CG doublets in codons whenever possible. Codon usage can benormalized against a plant preferred codon usage table (such as Table 3)and the G+C content preferably adjusted to about 50%. The resultingsequence should be examined to ensure that there are minimal putativeplant polyadenylation signals and ATTTA sequences.

TABLE 3 PREFERRED CODON USAGE IN PLANTS Amino Acid Codon Percent Usagein Plants ARG CGA 7 CGC 11 CGG 5 CGU 25 AGA 29 AGG 23 SER UCA 14 UCC 26UCG 3 UCU 21 AGC 21 AGU 15 THR ACA 21 ACC 41 ACG 7 ACU 31 PRO CCA 45 CCC19 CCG 9 CCU 26 HIS CAC 65 CAU 35 GLU GAA 48 GAG 52 ASP GAC 48 GAU 52TYR UAC 68 UAU 32 GYS UGC 78 UGU 22 LEU CUA 8 CUC 20 CUG 10 CUU 28 UUA 5UUG 30 ALA GCA 23 GCC 32 GCG 3 GCU 41 GLY GGA 32 GGC 20 GGG 11 GGU 37ILE AUA 12 AUC 45 AUU 43 VAL GUA 9 GUC 20 GUG 28 GUU 43 LYS AAA 36 AAG64 ASN AAC 72 AAU 28 GLN CAA 64 CAG 36 PHE UUC 56 UUU 44 MET AUG 100 TRPUGG 100

Restriction sites found in commonly used cloning vectors are alsopreferably avoided. However, placement of several unique restrictionsites throughout the gene is useful for analysis of gene expression orconstruction of gene variants.

5.8.7 “Plantized” Gene Constructs

The expression of a plant gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3′non-translated region which adds polyadenylate nucleotides to the 3′ endof the RNA. Transcription of DNA into mRNA is regulated by a region ofDNA usually referred to as the “promoter.” The promoter region containsa sequence of bases that signals RNA polymerase to associate with theDNA and to initiate the transcription of mRNA using one of the DNAstrands as a template to make a corresponding strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. These include the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of A. tumefaciens), the Cauliflower Mosaic Virus(CaMV) 19S and 35S promoters, the light-inducible promoter from thesmall subunit of ribulose bis-phosphate carboxylase (ssRUBISCO, a veryabundant plant polypeptide) and the mannopine synthase (MAS) promoter(Velten et al., 1984; Velten and Schell, 1985). All of these promotershave been used to create various types of DNA constructs which have beenexpressed in plants (see e.g., Intl. Pat. Appl. Publ. Ser. No. WO84/02913).

Promoters which are known or are found to cause transcription of RNA inplant cells can be used in the present invention. Such promoters may beobtained from plants or plant viruses and include, but are not limitedto, the CaMV35S promoter and promoters isolated from plant genes such asssRUBISCO genes. As described below, it is preferred that the particularpromoter selected should be capable of causing sufficient expression toresult in the production of an effective amount of protein.

The promoters used in the DNA constructs (i.e. chimeric plant genes) ofthe present invention may be modified, if desired, to affect theircontrol characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression.

The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNA's, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs, as presented in thefollowing examples. Rather, the non-translated leader sequence can bepart of the 5′ end of the non-translated region of the coding sequencefor the virus coat protein, or part of the promoter sequence, or can bederived from an unrelated promoter or coding sequence. In any case, itis preferred that the sequence flanking the initiation site conform tothe translational consensus sequence rules for enhanced translationinitiation reported by Kozak (1984).

The cry DNA constructs of the present invention may also contain one ormore modified or fully-synthetic structural coding sequences which havebeen changed to enhance the performance of the cry gene in plants. Thestructural genes of the present invention may optionally encode a fusionprotein comprising an amino-terminal chloroplast transit peptide orsecretory signal sequence.

The DNA construct also contains a 3′ non-translated region. The 3′non-translated region contains a polyadenylation signal which functionsin plants to cause the addition of polyadenylate nucleotides to the 3′end of the viral RNA. Examples of suitable 3′ regions are (1) the 3′transcribed, non-translated regions containing the polyadenylationsignal of Agrobacterium tumor-inducing (Ti) plasmid genes, such as thenopaline synthase (NOS) gene, and (2) plant genes like the soybeanstorage protein (7S) genes and the small subunit of the RuBP carboxylase(E9) gene.

5.9 Methods for Producing Insect-Resistant Transgenic Plants

By transforming a suitable host cell, such as a plant cell, with arecombinant cryET31, cryET40, cryET43, cryET44, cryET45, cryET46,cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET56, cryET57,cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66, cryET67,cryET68, cryET72, cryET73, and cryET83 gene-containing segment, theexpression of the encoded crystal protein (i.e., a bacterial crystalprotein or polypeptide having insecticidal activity againstcoleopterans) can result in the formation of insect-resistant plants.

By way of example, one may utilize an expression vector containing acoding region for a B. thuringiensis crystal protein and an appropriateselectable marker to transform a suspension of embryonic plant cells,such as wheat or corn cells using a method such as particle bombardment(Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated onmicroprojectiles into the recipient cells. Transgenic plants are thenregenerated from transformed embryonic calli that express theinsecticidal proteins.

The formation of transgenic plants may also be accomplished using othermethods of cell transformation which are known in the art such asAgrobacterium-mediated DNA transfer (Fraley et al., 1983).Alternatively, DNA can be introduced into plants by direct DNA transferinto pollen (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), byinjection of the DNA into reproductive organs of a plant (Pena et al.,1987), or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos (Neuhaus et al., 1987;Benbrook et al., 1986).

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, 1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983).

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

A transgenic plant of this invention thus has an increased amount of acoding region (e.g., a cryET31, cryET40, cryET43, cryET44, cryET45,cryET46, cryET47, cryET49, cryET51, cryET52, cryET53, cryET54, cryET56,cryET57, cryET59, cryET60, cryET61, cryET62, cryET63, cryET64, cryET66,cryET67, cryET68, cryET72, cryET73, and cryET83 gene) that encodes oneor more CryET31, CryET40, CryET43, CryET44, CryET45, CryET46, CryET47,CryET49, CryET51, CryET52, CryET53, CryET54, CryET56, CryET57, CryET59,CryET60, CryET61, CryET62, CryET63, CryET64, CryET66, CryET67, CryET68,CryET72, CryET73, and CryET83 polypeptides. A preferred transgenic plantis an independent segregant and can transmit that gene and its activityto its progeny. A more preferred transgenic plant is homozygous for thatgene, and transmits that gene to all of its offspring on sexual mating.Seed from a transgenic plant may be grown in the field or greenhouse,and resulting sexually mature transgenic plants are self-pollinated togenerate true breeding plants. The progeny from these plants become truebreeding lines that are evaluated for, by way of example, increasedinsecticidal capacity against coleopteran insects, preferably in thefield, under a range of environmental conditions. The inventorscontemplate that the present invention will find particular utility inthe creation of transgenic plants of commercial interest includingvarious turf grasses, wheat, corn, rice, barley, oats, a variety ofornamental plants and vegetables, as well as a number of nut- andfruit-bearing trees and plants.

5.10 Definitions

The following words and phrases have the meanings set forth below.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Identity or percent identity: refers to the degree of similarity betweentwo nucleic acid or protein sequences. An alignment of the two sequencesis performed by a suitable computer program. A widely used and acceptedcomputer program for performing sequence alignments is CLUSTALW v1.6(Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number ofmatching bases or amino acids is divided by the total number of bases oramino acids, and multiplied by 100 to obtain a percent identity. Forexample, if two 580 base pair sequences had 145 matched bases, theywould be 25 percent identical. If the two compared sequences are ofdifferent lengths, the number of matches is divided by the shorter ofthe two lengths. For example, if there were 100 matched amino acidsbetween 200 and a 400 amino acid proteins, they are 50 percent identicalwith respect to the shorter sequence. If the shorter sequence is lessthan 150 bases or 50 amino acids in length, the number of matches aredivided by 150 (for nucleic acid bases) or 50 (for amino acids), andmultiplied by 100 to obtain a percent identity.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast or explant).

Structural gene: A polynucleotide sequence that encodes a polypeptide,that is expressed to produce a polypeptide, or which is cryptic orincapable of expression in its natural host cell but which can beisolated and purified and operably linked to at least a promoterfunctional in one or more host cell types to express the encodedpolypeptide.

Transformation: A process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication.

Transformed cell: A cell whose DNA has been altered by the introductionof an exogenous DNA molecule into that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell. Exemplary transgenic cells includeplant calli derived from a transformed plant cell and particular cellssuch as leaf, root, stem, e.g., somatic cells, or reproductive (germ)cells obtained from a transgenic plant.

Transgenic plant: A plant or progeny thereof derived from a transformedplant cell or protoplast, wherein the plant DNA contains an introducedexogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callus obtained from a transformed plant cell orprotoplast as being a transgenic plant, and that usage will be followedherein.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

5.11 Isolating Homologous Gene and Gene Fragments

The genes and δ-endotoxins according to the subject invention includenot only the full length sequences disclosed herein but also fragmentsof these sequences, or fusion proteins, which retain the characteristicinsecticidal activity of the sequences specifically exemplified herein.

It should be apparent to a person skill in this art that insecticidalδ-endotoxins can be identified and obtained through several means. Thespecific genes, or portions thereof, may be obtained from a culturedepository, or constructed synthetically, for example, by use of a genemachine. Variations of these genes may be readily constructed usingstandard techniques for making point mutations. Also, fragments of thesegenes can be made using commercially available exonucleases orendonucleases according to standard procedures. For example, enzymessuch as Bal31 or site-directed mutagenesis can be used to systematicallycut off nucleotides from the ends of these genes. Also, genes which codefor active fragments may be obtained using a variety of otherrestriction enzymes. Proteases may be used to directly obtain activefragments of these δ-endotoxins.

Equivalent δ-endotoxins and/or genes encoding these equivalentδ-endotoxins can also be isolated from Bacillus strains and/or DNAlibraries using the teachings provided herein. For example, antibodiesto the δ-endotoxins disclosed and claimed herein can be used to identifyand isolate other δ-endotoxins from a mixture of proteins. Specifically,antibodies may be raised to the portions of the δ-endotoxins which aremost constant and most distinct from other B. thuringiensisδ-endotoxins. These antibodies can then be used to specifically identifyequivalent δ-endotoxins with the characteristic insecticidal activity byimmunoprecipitation, enzyme linked immunoassay (ELISA), or Westernblotting.

A further method for identifying the δ-endotoxins and genes of thesubject invention is through the use of oligonucleotide probes. Theseprobes are nucleotide sequences having a detectable label. As is wellknown in the art, if the probe molecule and nucleic acid samplehybridize by forming a strong bond between the two molecules, it can bereasonably assumed that the probe and sample are essentially identical.The probe's detectable label provides a means for determining in a knownmanner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying formicidal δ-endotoxin genes ofthe subject invention.

The nucleotide segments which are used as probes according to theinvention can be synthesized by use of DNA synthesizers using standardprocedures. In the use of the nucleotide segments as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³²P, ¹²⁵I, ³⁵S, or the like. A probelabeled with a radioactive isotope can be constructed from a nucleotidesequence complementary to the DNA sample by a conventional nicktranslation reaction, using a DNase and DNA polymerase. The probe andsample can then be combined in a hybridization buffer solution and heldat an appropriate temperature until annealing occurs. Thereafter, themembrane is washed free of extraneous materials, leaving the sample andbound probe molecules typically detected and quantified byautoradiography and/or liquid scintillation counting.

Non-radioactive labels include, for example, ligands such as biotin orthyroxine, as well as enzymes such as hydrolases or peroxidases, or thevarious chemiluminescers such as luciferin, or fluorescent compoundslike fluorescein and its derivatives. The probe may also be labeled atboth ends with different types of labels for ease of separation, as, forexample, by using an isotopic label at the end mentioned above and abiotin label at the other end.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probes of thesubject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, by methods currently known to anordinarily skilled artisan, and perhaps by other methods which maybecome known in the future.

The potential variations in the probes listed is due, in part, to theredundancy of the genetic code. Because of the redundancy of the geneticcode, i.e., more than one coding nucleotide triplet (codon) can be usedfor most of the amino acids used to make proteins. Therefore differentnucleotide sequences can code for a particular amino acid. Thus, theamino acid sequences of the B. thuringiensis δ-endotoxins and peptidescan be prepared by equivalent nucleotide sequences encoding the sameamino acid sequence of the protein or peptide. Accordingly, the subjectinvention includes such equivalent nucleotide sequences. Also, inverseor complement sequences are an aspect of the subject invention and canbe readily used by a person skilled in this art. In addition it has beenshown that proteins of identified structure and function may beconstructed by changing the amino acid sequence if such changes do notalter the protein secondary structure (Kaiser and Kezdy, 1984). Thus,the subject invention includes mutants of the amino acid sequencedepicted herein which do not alter the protein secondary structure, orif the structure is altered, the biological activity is substantiallyretained. Further, the invention also includes mutants of organismshosting all or part of a δ-endotoxin encoding a gene of the invention.Such mutants can be made by techniques well known to persons skilled inthe art. For example, UV irradiation can be used to prepare mutants ofhost organisms. Likewise, such mutants may include asporogenous hostcells which also can be prepared by procedures well known in the art.

6.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

6.1 Example 1

Identification of B. thuringiensis Strains Containing Novel δ-Endotoxins

Wild-type B. thuringiensis strains containing novel insecticidal proteingenes were identified by Southern blot hybridization studies employingspecific DNA probes. Twenty-four unique cry genes were discovered thatare related to B. thuringiensis genes in the cry1, cry2, or cry9 classesof toxin genes.

Various methods were employed to clone the novel genes and express themin a crystal protein-negative (Cry−) strain of B. thuringiensis. Thesemethods include PCR™ amplification of the region of cry1-related genesthat encodes the active portion of the toxin gene. The PCR™ product isthen joined to a fragment from the cry1Ac gene encoding the C-terminalregion of the protoxin. This gene fusion was then expressed in a B.thuringiensis recombinant strain to produce a hybrid protoxin. In thisinstance, it is recognized that the sequence of the amplified DNA can beused to design hybridization probes to isolate the entire codingsequence of the novel cry gene from the wild-type B. thuringiensisstrain.

Wild-type B. thuringiensis strains were screened in a bioassay toidentify strains that are toxic to larvae of lepidopteran insects(procedure described in Example 10). Active strains were then examinedgenetically to determine if they contain novel toxin genes. The methodused to make this determination is described below and includesisolation of genomic DNA from the B. thuringiensis strain, restrictionenzyme digestion, Southern blot hybridization, and analysis of thehybridizing restriction fragments to determine which genes are presentin a strain.

Total genomic DNA was extracted by the following procedure. Vegetativecells were resuspended in a lysis buffer containing 50 mM glucose, 25 mMTris-HCl (pH 8.0), 10 mM EDTA, and 4 mg/ml lysozyme. The suspension wasincubated at 37° C. for 1 h. Following incubation, the suspension wasextracted once with an equal volume of phenol, then once with an equalvolume of phenol:chloroform:isoamyl alcohol (50:48:2), and once with anequal volume of chloroform:isoamyl alcohol (24:1). The DNA wasprecipitated from the aqueous phase by the addition of one-tenth volume3 M sodium acetate and two volumes of 100% ethanol. The precipitated DNAwas collected by centrifugation, washed with 70% ethanol and resuspendedin distilled water.

The DNA samples were digested with the restriction enzymes ClaI andPstI. The combination of these two enzymes give a digestion pattern offragments that, when hybridized with the probe wd207 (described below),allows the identification of many of the known cry1-related toxin genes.Hybridizing fragments that did not correspond to the fragment sizesexpected for the known genes were classified as unknown and werecandidates for cloning and characterization.

The digested DNA was size fractionated by electrophoresis through a 1.0%agarose gel in 1×TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 MEDTA) overnight at 2 V/cm of gel length. The fractionated DNA fragmentswere then transferred to a Millipore Immobilon-NC® nitrocellulose filter(Millipore Corp., Bedford, Mass.) according to the method of Southern(1975). The DNA fragments were fixed to the nitrocellulose by baking thefilter at 80° C. in a vacuum oven.

To identify the DNA fragment(s) containing the sequences related to cry1genes, the oligonucleotide wd207 was radioactively labeled at the 5′ endand used as a hybridization probe. To radioactively label the probe, 1-5pmoles of wd207 were added to a reaction (20 ul total volume) containing3 ul [γ-³²P]ATP (3,000 Ci/mmole at 10 mCi/ml), 70 mM Tris-HCl, pH 7.8,10 mM MgCl₂, 5 mM DTT, and 10 units T4 polynucleotide kinase (PromegaCorp., Madison, Wis.). The reaction was incubated for 20 min at 37° C.to allow the transfer of the radioactive phosphate to the 5′-end of theoligonucleotide, thus making it useful as a hybridization probe.

The oligonucleotide probe used in this analysis, designated wd207, hasthe following sequence:

(SEQ ID NO:51) 5′-TGGATACTTGATCAATATGATAATCCGTCACATCTGTTTTTA-3′

This oligonucleotide was designed to specifically hybridize to aconserved region of cry1 genes downstream from the proteolyticactivation site in the protoxin. Table 4 lists some of the B.thuringiensis toxin genes and their identities with wd207. Theorientation of the wd207 sequence is inverted and reversed relative tothe coding sequences of the cry genes.

TABLE 4 cry Gene % Identity to wd207 Nucleotide Position in CDS cry1Aa 100% 1903-1944 cry1Ba 95.2% 1991-2032 cry1Ca 97.6% 1930-1971 cry1Da97.6% 1858-1899 cry1Ea 97.6% 1885-1926

The labeled probe was then incubated with the nitrocellulose filterovernight at 45° C. in 3×SSC (1×SSC=0.15 M NaCl, 0.015 M sodiumcitrate), 0.1% SDS, 10×Denhardt's reagent (0.2% BSA, 0.2%polyvinylpyrrolidone, 0.2% Ficoll), and 0.2 mg/ml heparin. Followingthis incubation period, the filter was washed in several changes of3×SSC, 0.1% SDS at 45° C. The filter was blotted dry and exposed toKodak X-OMAT AR X-ray film (Eastman Kodak Co., Rochester, N.Y.)overnight at −70° C. with an intensifying screen to obtain anautoradiogram.

The autoradiograms were analyzed to determine which wild-type B.thuringiensis strains contained cry1 genes that could be novel. Sincethe probe was only 42 nucleotides, it is unlikely that recognition sitesfor the restriction endonucleases ClaI and PstI would occur within thehybridizing region of the cry1-related genes. Therefore, it was assumedthat each hybridizing restriction fragment represented one cry1-relatedgene. The sizes, in kilobases (kb), of the hybridizing restrictionfragments were determined based on the migration of the fragment in theagarose gel relative to DNA fragments of known size. The size of afragment could be used to determine if that fragment represented a knowncry1 gene. For example, from the DNA sequence of the cry1Ac gene it wasknown that wd207 would hybridize to a 0.43 kb fragment after digestionof cry1Ac DNA with ClaI and PstI. If the Southern blot analysis of astrain showed a 0.43 kb hybridizing fragment, that strain was assigned aprobable genotype of cry1Ac. Fragments that could not be easily assigneda probable genotype were selected as candidates for further analysis.Because many cry1-containing strains have more than one cry1-relatedgene, all fragments were given a putative designation.

TABLE 5 SUMMARY OF GENES AND PROTEINS Polypeptide PolypeptidePolynucleotide WT- Recomb. Gene Cloning DNA Cloning Designation Seq. IDNo.: Seq ID No.: Strain Strain Family Method¹ Probe² Vector Plasmid CryET31 2 1 EG6701 EG11562 cry2 MboI cry2a pHT315 pEG1331 Cry ET40 4 3EG5476 EG11901 cry1 PCR ™ — pEG1064 pEG1901 Cry ET43 6 5 EG2878 EG7692cry1 PCR ™ — pEG1064 pEG1806 Cry ET44 8 7 EG3114 EG11629 cry1 PCR ™ —pEG1064 pEG1807 Cry ET45 10 9 EG3114 EG7694 cry1 PCR ™ — pEG1064 pEG1808Cry ET46 12 11 EG6451 EG7695 cry1 PCR ™ — pEG1064 pEG1809 Cry ET47 14 13EG6451 EG7696 cry1 PCR ™ — pEG1064 pEG1810 Cry ET49 16 15 EG6451 EG11630cry1 PCR ™ — pEG1064 pEG1812 Cry ET51 18 17 EG5391 EG11921 cry1 MboIwd207 pHT315 pEG1912 Cry ET52 20 19 EG10475 EG11584 cry1 BamHI wd207pEG290 pEG1340 Cry ET53 22 21 EG3874 EG11906 cry1 MboI cry1Aa pHT315pEG1904 Cry ET54 EG3874 EG11907 cry1 MboI cry1Aa pHT315 pEG1905 Cry ET5624 23 EG3874 EG11909 cry1 MboI cry1Aa pHT315 pEG1907 Cry ET57 26 25EG3874 EG11910 cry1 MboI cry1Aa pHT315 pEG1908 Cry ET59 28 27 EG9290EG12102 cry9 MboI pr56, pHT315 pEG945 cryET59 Cry ET60 30 29 EG9290EG12103 cry9 MboI pr56, pHT315 pEG946 cryET59 Cry ET61 32 31 EG4612EG11634 cry1 MboI wd207 pHT315 pEG1813 Cry ET62 34 33 EG6831 EG11635cry1 MboI wd207 pHT315 pEG1814 Cry ET63 36 35 EG4623 EG11636 cry1 MboIwd207 pHT315 pEG1815 Cry ET64 38 37 EG4612 EG11638 cry1 MboI wd207pHT315 pEG1816 Cry ET66 40 39 EG5020 EG11640 cry1 MboI wd207 pHT315pEG1817 Cry ET67 42 41 EG4869 EG11642 cry1 MboI wd207 pHT315 pEG1818 CryET68 44 43 EG5020 EG11644 cry1 MboI wd207 pHT315 pEG1819 Cry ET72 46 45EG4420 EG11440 cry2 HindIII cry2Aa pEG597 pEG1260 Cry ET73 48 47 EG3874EG11465 cry2 HindIII cry2Aa pEG597 pEG1279 Cry ET83 50 49 EG6346 EG11785cry9 MboI cryET59, pHT315 pEG397 cryET83 ¹Methods include theconstruction of genomic libraries containing partial MboI fragments(Example 4), the construction of genomic libraries containingsize-selected BamHI or HindIII restriction fragments (Example 5), theamplification of novel cry sequences by PCR ™ and the construction ofnovel cry gene fusions (Example 6). ²Hybridization probes included the700 base pair EcoRI fragment obtained from digestion of the cry1Aa gene,gene fragments from the cry2Aa, cryET59, and cryET83 genes, andsynthetic oligonucleotides (wd207, pr56).

6.2 Example 2

Identification of B. thuringiensis Strains Containing Novel Cry2-RelatedGenes

Proteins encoded by the cry2 class of B. thuringiensis class of toxingenes have activity on the larvae of lepidopteran and diopteran insects.Southern blot hybridization analysis of DNA extracted fromlepidopteran-active strains was utilized to identify novel cry2-relatedgenes. Total genomic DNA was isolated as described in Section 6.1. TheDNA was digested with the restriction endonuclease Sau3A and run on a1.2% agarose gel as described. The digested DNA was transferred tonitrocellulose filters to be probed with a DNA fragment containing thecry2Aa gene. Hybridizations were performed at 55° C. and the filterswashed and exposed to X-ray film to obtain an autoradiogram.

Sau3A digestion followed by hybridization with the cry2Aa gene gavecharacteristic patterns of hybridizing fragments allowing theidentification of the cry2Aa, cry2Ab, and cry2Ac genes. Hybridizingfragments that differed from these patterns indicated the presence of anovel cry2-related gene in that strain.

Once a strain was identified as containing one or more novelcry2-related genes, an additional Southern blot hybridization wasperformed. The procedures were the same as those already describedabove, except another restriction enzyme, usually HindIII, was used.Since an enzyme like HindIII (a “six base cutter”) cuts DNA lessfrequently than does Sau3A or MboI, it was more likely to generate arestriction fragment containing the entire cry2-related gene which couldthen be readily cloned.

6.3 Example 3

Identification of B. thuringiensis Strains Containing Novel Cry9-TypeGenes

A cry9-specific oligonucleotide, designated pr56, was designed tofacilitate the identification of strains harboring cry9-type genes. Thisoligonucleotide corresponds to nucleotides 4349-4416 of the gene(GenBank Accession No. Z37527). The sequence of pr56 was as follows:

(SEQ ID NO:52) 5′-AGTAACGGTGTTACTATTAGCGAGGGCGGTCCATTCTTTAAAGGTCGTGCACTTCAGTTAGC-3′.

B. thuringiensis isolates were spotted or “patched” on SGNB plates, withno more than 50 isolates per plate, and grown overnight at 25° C. The B.thuringiensis colonies were transferred to nitrocellulose filters andthe filters placed, colony side up, on fresh SGNB plates for overnightgrowth at 30° C. Subsequently, the filters were placed, colony side up,on Whatman paper soaked in denaturing solution (1.5 M NaCl, 0.5 N NaOH)for 20 min. After denaturation, the filters were placed on Whatman papersoaked in neutralizing solution (3 M NaCl, 1.5 M Tris-HCl, pH 7.0) for20 min. Finally, the filters were washed in 3×SSC (1×SSC=0.15 M NaCl and0.015 M sodium citrate) to remove cellular debris and baked in a vacuumoven at 80° C. for 90 min.

The cry9-specific oligonucleotide pr56 (˜10 pmoles) was end-labeled with[γ-³²P]ATP using T4 polynucleotide kinase. The labeling reaction wascarried out at 37° C. for 20 min and terminated by incubating thereaction at 100 C for 3 min. After ethanol precipitation, the labeledoligonucleotide was resuspended in 100 μl distilled H₂O.

The filters were incubated with the cry9-specific probe in 6×SSC,10×Denhardt's solution, 0.5% glycine, 0.2% SDS at 47° C. overnight. Thefilters were washed twice in 3×SSC, 0.1% SDS for 15 min at 47° C. andtwice in 1×SSC, 0.1% SDS for 15 min at 47° C. The dried filters wereexposed to X-OMAT XAR-5 film (Eastman Kodak Co.) at −70° C. using anintensifying screen. The developed autoradiogram revealed 24 isolates ofB. thuringiensis containing DNA that hybridized to the cry9 probe.

To identify cry9C-type genes among these strains, two opposingoligonucleotide primers specific for the cry9C gene (GenBank AccessionNo. Z37527) were designed for polymerase chain reaction (PCR™) analyses.The sequence of pr58 is:

5′-CGACTTCTCCTGCTAATGGAGG-3′. (SEQ ID NO:53)The sequence of pr59 is:

5′-CTCGCTAATAGTAACACCGTTACTTGCC-3′. (SEQ ID NO:54)Plasmid DNAs were isolated from the isolates of B. thuringiensisbelieved to contain cry9-type genes. B. thuringiensis isolates weregrown overnight at 30° C. on Luria agar plates and 2 loopfuls of cellsfrom each isolate were suspended in 50 mM glucose, 10 mM Tris-HCl, 1 mMEDTA (1×GTE) containing 4 mg/ml lysozyme. After a 10 min incubation atroom temperature, plasmid DNAs were extracted using a standard alkalinelysis procedure (Maniatis et al., 1982). The plasmid DNAs wereresuspended in 20 μl of 1×TE (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Twomicroliters of the plasmid DNA preparations were used in the PCR™reactions. Amplifications were performed in 100 μl volumes with aPerkin-Elmer DNA Thermocycler (Perkin-Elmer Cetus, Foster City, Calif.)using materials and methods provided in the Perkin-Elmer GeneAmp™ kit.Conditions for the PCR™ were as follows: 95° C. for 30 sec, 46° C. for30 sec, 70° C. for 1 min; 30 cycles. A PCR™ using these primers and thecry9C gene as a template should yield a DNA fragment of ˜970 bp. Oftwenty-four strains found to hybridize to the cry9 probe (SEQ ID NO:XX),only one strain, EG9290, yielded the predicted amplified DNA fragment.

6.4 Example 4

Cloning of B. thuringiensis Toxin Genes by Constructing MboI PartialDigest Libraries

The restriction endonuclease MboI was utilized in the construction ofgenomic DNA libraries because it has a recognition sequence of four basepairs which occurs frequently in long stretches of DNA. Total genomicDNA was isolated from B. thuringiensis strains as described in Section6.1. The DNA was digested under conditions allowing limited cleavage ofa DNA strand. The method of establishing these conditions has beendescribed (Maniatis et al, 1982). Digestion of DNA in this mannercreated a set of essentially randomly cleaved, overlapping fragmentswhich were used to create a library representative of the entire genome.

The digested DNA fragments were separated, according to size, by agarosegel electrophoresis through a 0.6% agarose, 1×TBE gel, overnight at 2volts/cm of gel length. The gel was stained with ethidium bromide sothat the digested DNA could be visualized when exposed to long-wave UVlight. A razor blade was used to excise a gel slice containing DNAfragments of approximately 9-kb to 12-kb in size. The DNA fragments wereremoved from the agarose by placing the slice in a dialysis bag withenough TE (10 mM Tris-HCl, 1 mM EDTA) to cover the slice. The bag wasthen closed and placed in a horizontal electrophoresis apparatus filledwith 1×TBE buffer. The DNA was electroeluted from the slice into the TEat 100 volts for 2 h. The TE was removed from the bag, extracted withphenol:chloroform (1:1), followed by extraction with chloroform. The DNAfragments are then collected by the standard technique of ethanolprecipitation (see Maniatis et al., 1982).

To create a library in E. coli of the partially-digested DNA, thefragments were ligated into the shuttle vector, pHT315 (Arantes andLereclus, 1991). This plasmid contains replication origins for E. coliand B. thuringiensis, genes for resistance to the antibioticserythromycin and ampicillin, and a multiple cloning site. The MboIfragments were mixed with BamHI-digested pHT315 that had been treatedwith calf intestinal, or bacterial, alkaline phosphatase (GibcoBRL,Gaithersburg, Md.) to remove the 5′-phosphates from the digestedplasmid, preventing re-ligation of the vector to itself. Afterpurification, T4 ligase and a ligation buffer (Promega Corp., Madison,Wis.) were added to the reaction containing the digested vector and theMboI fragments. These were incubated overnight at 15° C., or at roomtemperature for 1 h, to allow the insertion and ligation of the MboIfragments into the pHT315 vector DNA.

The ligation mixture was then introduced into transformation-competentE. coli SURE® cells (Stratagene Cloning Systems, La Jolla, Calif.),following procedures described by the manufacturer. The transformed E.coli cells were then plated on LB agar plates containing 50-75 μg/mlampicillin and incubated overnight at 37° C. The growth of severalhundred ampicillin-resistant colonies on each plate indicated thepresence of recombinant plasmid in the cells of each of those colonies.

To isolate the colonies harboring sequences encoding toxin genes, thecolonies were first transferred to nitrocellulose filters. This wasaccomplished by simply placing a circular nitrocellulose filter(Millipore HATF 08525, Millipore Corp., Bedford, Mass.) directly on topof the LB-ampicillin agar plates containing the transformed colonies.When the filter was slowly peeled off of the plate the colonies stick tothe filter giving an exact replica of the pattern of colonies from theoriginal plate. Enough cells from each colony were left on the platethat 5 to 6 h of growth at 37° C. restored the colonies. The plates werethen stored at 4° C. until needed. The nitrocellulose filters with thetransferred colonies are then placed, colony-side up, on freshLB-ampicillin agar plates and allowed to grow at 37° C. until theyreached an approximate 1 mm diameter.

To release the DNA from the recombinant E. coli cells the nitrocellulosefilters were placed, colony-side up, on 2-sheets of Whatman 3MMchromatography paper (Whatman International Ltd., Maidstone, England)soaked with 0.5 N NaOH, 1.5 M NaCl for 15 min. This treatment lysed thecells and denatured the released DNA allowing it to stick to thenitrocellulose filter. The filters were then neutralized by placing thefilters, colony-side up, on 2 sheets of Whatman paper soaked with 1 MNH₄-acetate, 0.02 M NaOH for 10 min. The filters were rinsed in 3×SSC,air dried, and baked for 1 h at 80° C. in a vacuum oven. The filterswere then ready for use in hybridization studies using probes toidentify different classes of B. thuringiensis genes, as described inthe above examples.

In order to identify colonies containing cloned cry1-related genes, thecry1-specific oligonucleotide wd207 was labeled at the 5′-end with[γ-³²P]ATP and T4 polynucleotide kinase. The labeled probe was added tothe filters in 3×SSC, 0.1% SDS, 10×Denhardt's reagent (0.2% BSA, 0.2%polyvinylpyrrolidone, 0.2% Ficoll), 0.2 mg/ml heparin and incubatedovernight at 47° C. These conditions allowed hybridization of thelabeled oligonucleotide to related sequences present on thenitrocellulose blots of the transformed E. coli colonies. Followingincubation the filters were washed in several changes of 3×SSC, 0.1% SDSat 45° C. The filters were blotted dry and exposed to Kodak X-OMAT ARX-ray film (Eastman Kodak Co., Rochester, N.Y.) overnight at −70° C.with an intensifying screen.

Colonies that contain cloned cry1-related sequences were identified byaligning signals on the autoradiogram with the colonies on the originaltransformation plates. The isolated colonies were then grown inLB-ampicillin liquid medium from which the cells could be harvested andrecombinant plasmid prepared by the standard alkaline-lysis miniprepprocedure (Maniatis et al., 1982). The plasmid DNA was then used as atemplate for DNA sequencing reactions necessary to confirm that thecloned gene was novel. If the cloned gene was novel, the plasmid wasthen introduced into a crystal protein-negative strain of B.thuringiensis (Cry) so that the encoded protein could be expressed andcharacterized. These procedures are described in detail in the followingsections.

6.5 Example 5

Cloning of Specific Endonuclease Restriction Fragments

The identification of a specific restriction fragment containing a novelB. thuringiensis gene has been described for cry2-related genes inSection 2. The procedure for cloning a restriction fragment of knownsize was essentially the same as described for cloning an MboI fragment.The DNA was digested with a restriction enzyme (e.g., HindIII), and runthrough an agarose gel to separate the fragments by size. Fragments ofthe proper size, identified by Southern blot analysis (Example 2), wereexcised with a razor blade and electroeluted from the gel slice into TEbuffer from which they could be precipitated. The isolated restrictionfragments were then ligated into an E. coli/B. thuringiensis shuttlevector and transformed into E. coli to construct a size-selectedlibrary. The library could then be hybridized with a specific geneprobe, as described in Example 4, to isolate the colony containing thecloned novel gene.

6.6 Example 6

Cloning of PCR™-Amplified Fragments

A rapid method for cloning and expressing novel cry1 gene fragments fromB. thuringiensis was developed using the polymerase chain reaction.Flanking primers were designed to anneal to conserved regions 5′ to andwithin cry1 genes. With the exception of certain cry3 genes, most B.thuringiensis cry genes are transcriptionally regulated, at least inpart, by RNA polymerases containing the mother cell-specific σ^(E) orsigE, sigma factor. These σ^(Y)-regulated cry genes possess 5′ promotersequences that are recognized by σ^(E). Alignment of these promotersequences reveals considerable sequence variation, although a consensussequence can be identified (Baum and Malvar, 1995). A primer, designated“sigE”, containing a sequence identical to the cry1Ac σ^(E) promotersequence, was designed that would anneal to related GE promotersequences 5′ to uncharacterized cry genes. The sigE primer also includesa BbuI site (isoschizimer: SphI) to facilitate cloning of amplifiedfragments. The sequence of the sigE primer is shown below:

(SEQ ID NO:55) 5′-ATTTAGTAGCATGCGTTGCACTTTGTGCATTTTTTCATAAGATGAGTCATATGTTTTAAAT-3′.

The opposing primer, designated KpnR, anneals to a 3′-proximal region ofthe cry1 gene that is generally conserved. This primer incorporates anAsp718 site (isoschizimer: KpnI) conserved among the cry1A genes tofacilitate cloning of the amplified fragment and to permit theconstruction of fusion proteins containing a carboxyl-terminal portionof the Cry1Ac protein. The sequence of the KpnR primer is shown below:

5′-GGATAGCACTCATCAAAGGTACC-3′ (SEQ ID NO:56)

PCR™s were carried out using a Perkin Elmer DNA thermocycler and thefollowing parameters: 94° C., 2 min.; 40 cycles consisting of 94° C., 30sec; 40° C., 2 min; 72° C., 3 min; and a 10 second extension added tothe 72° C. incubation after 20 cycles. The standard PCR™ buffer (100 μlvolume) was modified to include 1×Taq Extender buffer, 25 μM each of thesigE and KpnR primers, and 0.5-1.0 μl of Taq Extender (Stratagene Inc.)in addition to 0.5-1.0 μl of Taq polymerase. Typically, 1-2 μl of theDNA preparations from novel B. thuringiensis isolates were included inthe PCR™s. PCR™s with cry genes incorporating these primers resulted inthe amplification of a ˜2.3-kb DNA fragment flanked by restriction sitesfor BbuI and Asp718.

For the cloning and expression of these gene fragments, the cry1Acshuttle vector pEG1064 was used. This plasmid is derived from the cry1Acshuttle vector pEG857 (Baum et al., 1990), with the followingmodifications. A frameshift mutation was generated at a unique NcoI sitewithin the cry1Ac coding region by cleaving pEG857 with the restrictionendonuclease NcoI, blunt-ending the NcoI-generated ends with Klenowpolymerase and ligating the blunt ends with T4 ligase. In similarfashion, an Asp718 site located in the multiple cloning site 3′ to thecry1Ac gene was removed, leaving only the single Asp718 site containedwithin the cry1Ac coding sequence. The resulting plasmid, pEG1064,cannot direct the production of crystal protein when introduced into anacrystalliferous (Cry⁻) strain of B. thuringiensis because of theframeshift mutation. For cloning and expression of unknown cry genes,pEG1064 was cleaved with BbuI and Asp718 and the vector fragmentpurified following gel electrophoresis. Amplified fragments of unknowncry genes, obtained by PCR™ amplification of total B. thuringiensis DNA,were digested with the restriction endonucleases BbuI and Asp718 andligated into the BbuI and Asp718 sites of the pEG1064 vector fragment.The ligation mixture was used to transform the Cry⁻ B. thuringiensisstrains, EG10368 or EG10650, to chloramphenicol resistance using anelectroporation protocol previously described (Mettus and Macaluso,1990) Chloramphenicol-resistant (Cm^(R)) isolates were evaluated forcrystal protein production by phase-contrast microscopy. Crystal forming(Cry+) isolates were subsequently grown in C2 liquid broth medium(Donovan et al., 1988) to obtain crystal protein for SDS-PAGE analysisand insect bioassay.

Because of the frameshift mutation within the cry1Ac gene, the crystalproteins obtained from the transformants could not be derived from thevector pEG1064. The Cry⁺ transformants thus contained unknown cry genefragments fused, at the Asp718 site, to a 3′-portion of the cry1Ac gene.Transcription of these gene fusions in B. thuringiensis was presumablydirected from the σ^(E) promoter incorporated into the amplified crygene fragment. The fusion proteins, containing the entire active toxinregion of the unknown Cry protein, were capable of producing crystals inB. thuringiensis.

6.7 Example 7

Cloning of Cry9-Related Genes

Total DNA was isolated from B. thuringiensis strain EG9290 for cloningstudies. EG9290 was grown overnight at 30° C. in 1× brain heartinfusion, 0.5% glycerol (BHIG). In the morning, 500 μl of the overnightgrowth was suspended in 50 ml BHIG and the culture incubated at 30° C.with agitation until the culture reached a Klett reading of 150 (redfilter). The cells were harvested by centrifugation, suspended in 5 ml1×GTE buffer containing 4 mg/ml lysozyme and 100 μg/ml Rnase A, andincubated at 37° C. for 20 min. The cells were lysed by the addition of0.5 ml of 20% SDS. The released DNA was precipitated by the addition of2.5 ml 7.5 M ammonium acetate and 7 ml of isopropanol. The precipitatedDNA was spooled out of the mixture using a glass micropipette and washedin 80% ethanol. The DNA was resuspended in 10 ml 1×TE, extracted withone volume each of buffered phenol and chloroform:isoamyl alcohol(24:1), and precipitated as before. The spooled DNA was washed in 80%ethanol, allowed to air dry for several min, and suspended in 600 μl1×TE. The DNA concentration was estimated at 500 μg/ml.

A library of EG9290 total DNA was constructed using partially digestedMboI fragments of EG9290 DNA and the general methods described herein.The partial MboI fragments were inserted into the unique BamHI site ofcloning vector pHT315. The ligation mixture was used to transform E.coli Sure™ cells to ampicillin resistance by electroporation employingelectrocompetent cells and protocols provided by Stratagene (La Jolla,Calif.) and the BioRad Gene Pulser™ apparatus (Bio-Rad Laboratories,Hercules, Calif.). Recombinant clones harboring cry9-type genes wereidentified by colony blot hybridization using a ³²P-labeled probeconsisting of the putative cry9C fragment generated by amplification ofEG9290 DNA with primers pr58 and pr59. Plasmid DNAs were extracted fromthe E. coli clones using a standard alkaline lysis procedure.

Plasmid DNAs from the E. coli recombinant clones were used to transformB. thuringiensis strain EG10368 to erythromycin resistance using theelectroporation procedure described by Mettus and Macaluso (1990). Cellswere plated onto starch agar plates containing 20 μg/ml erythromycin andincubated at 30° C. After six days, colonies with a more opaqueappearance were recovered from the plates and streaked out onto freshstarch agar plates containing 20 μg/ml erythromycin to isolate singlecolonies. Colonies exhibiting a more opaque appearance were observed toproduce large parasporal inclusions/crystals by phase-contrastmicroscopy.

Recombinant EG10368 clones producing parasporal inclusion/crystals wereevaluated for crystal protein production in broth culture. Singlecolonies were inoculated into C2 medium containing 10 μg/ml erythromycinand grown at 30° C. for 3 days at 28-30° C., at which time the cultureswere fully sporulated and lysed. Spores and crystals were pelleted bycentrifugation and resuspended in 20 mM Tris-HCl, 1 mM EDTA, pH 7.0.Aliquots of this material were analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). Two EG10368 recombinant clones, initiallyidentified as 9290-2 and 9290-3, were observed to produce distinctproteins of 130 kDa. 9290-2 was designated EG12102 and 9290-3 wasdesignated EG12103. The EG12102 protein was designated CryET59 while theEG12103 protein was designated CryET60.

Plasmid DNAs were prepared from EG12102 and EG12103 using a standardalkaline lysis procedure. Digestion of the plasmids with the restrictionendonuclease XbaI confirmed that the two strains harbored distinct crygenes. The cry plasmids of EG12102 and EG12103, designated pEG945 andpEG946, respectively, were used to transform E. coli Sure™ cells toampicillin resistance by electroporation, employing electrocompetentcells and protocols provided by Stratagene Inc. The E. coli recombinantstrain containing pEG945 was designated EG12132, and the E. colirecombinant strain containing pEG946 was designated EG12133. pEG945 andpEG946 were purified from the E. coli recombinant strains using theQIAGEN midi-column plasmid purification kit and protocols (QIAGEN Inc.,Valencia, Calif.).

The cryET83 gene was cloned from B. thuringiensis strain EG6346subspecies aizawai using similar methods. Southern blot analysis ofgenomic DNA from EG6346 revealed a unique restriction fragment thathybridized to the cryET59 probe. A series of degenerate oligonucleotideprimers, pr95, pr97, and pr98, were designed to amplify cry9-relatedsequences from genomic DNA. The sequences of these primers are as shown:

pr95: (SEQ ID NO:57) 5′-GTWTGGACSCRTCGHGATGTGG-3′ pr97: (SEQ ID NO:58)5′-TAATTTCTGCTAGCCCWATTTCTGGATTTAATTGTTGATC-3′ pr98: (SEQ ID NO:59)5′-ATWACNCAAMTWCCDTTRG -3′where D=A, G; H=A, C, T; M=A, C; N=A, C, G, T; R=A, G; S═C, G; and W=A,T.

A PCR™ using Taq polymerase, Taq Extender™ (Stratagene, La Jolla,Calif.), the opposing primers pr95 and pr97, and total EG6346 DNAyielded a DNA fragment that was faintly visible on an ethidiumbromide-stained agarose gel. This DNA served as the template for asecond round of PCR™ using the opposing primers pr97 and pr98. Theresulting amplified DNA fragment was suitable for cloning and served asa hybridization probe for subsequent cloning experiments. A library ofEG6346 total DNA was constructed using partially digested 9-12 kb MboIfragments of EG6346 DNA ligated into the unique BamHI site of cloningvector pHT315. E. coli recombinant clones harboring the cryET83 genewere identified by colony blot hybridization using the EG6346-specificDNA fragment as a chemiluminescent hybridization probe and the CDP-Star™nucleic acid chemiluminescent reagent kit from NEN™ Life ScienceProducts (Boston, Mass.) to prepare the hybridization probe. Therecombinant plasmid harboring the cryET83 gene was designated pEG397.The E. coli recombinant stain containing pEG397 was designated EG11786.The B. thuringiensis recombinant strain containing pEG397 was designatedEG11785.

6.8 Example 8

Sequencing of Cloned B. thuringiensis Toxin Genes

Partial sequences for the cloned toxin genes were determined followingestablished dideoxy chain-termination DNA sequencing procedures (Sangeret al., 1977). Preparation of the Double Stranded Plasmid Template DNAwas Accomplished Using a standard alkaline lysis procedure or using aQIAGEN plasmid purification kit (QIAGEN Inc., Valencia, Calif.). Thesequencing reactions were performed using the Sequenase™ Version 2.0 DNASequencing Kit (United States Biochemical/Amersham Life Science Inc.,Cleveland, Ohio) following the manufacturer's procedures and using³⁵S-dATP as the labeling isotope (obtained from DuPont NEN® ResearchProducts, Boston, Mass.). Denaturing gel electrophoresis of thereactions is done on a 6% (wt./vol.) acrylamide, 42% (wt./vol.) ureasequencing gel. The dried gels are exposed to Kodak X-OMAT AR X-ray film(Eastman Kodak Company, Rochester, N.Y.) overnight at room temperature.Alternatively, some cry genes were sequenced using automated sequencingmethods. DNA samples were sequenced using the ABI PRISM™ DyeDeoxysequencing chemistry kit (Applied Biosystems, Foster City, Calif.)according to the manufacturer's suggested protocol. The completedreactions were run on as ABI 377 automated DNA sequencer. DNA sequencedata were analyzed using Sequencher™ v3.0 DNA analysis software (GeneCodes Corp., Ann Arbor, Mich.). Successive oligonucleotides to be usedfor priming sequencing reactions were designed from the sequencing dataof the previous set of reactions.

The sequence determination for the cry1-related genes involved the useof the oligonucleotide probe wd207, described in Example 2, as theinitial sequencing primer. This oligonucleotide anneals to a conservedregion of cry1 genes, but because of the inverted and reversedorientation of wd207, it generates sequence towards the 5′-end of thecoding region allowing sequence of the variable region of the gene to beread. A typical sequencing run of 250-300 nucleotides was usuallysufficient to determine the identity of the gene. If additional datawere necessary, one or more additional oligonucleotides could besynthesized to continue the sequence until it could be determined if thesequence was unique. In cases where wd207 did not function well as aprimer, other oligonucleotides, designed to anneal to conserved regionsof cry1 genes, were used. One such oligonucleotide was the KpnR primerdescribed herein above.

The sequencing of the cloned cry2-related genes followed the samegeneral procedures as those described for the cry1 genes, except thatoligonucleotides specific for conserved regions in cry2 genes were usedas sequencing primers. The two primers used in these examples were wd268and wd269, shown below.

Primer wd268 corresponds to cry2Aa nucleotides 579-5975′-AATGCAGATGAATGGGG-3′. (SEQ ID NO:60) Primer wd269 corresponds tocry2Aa 1740-1757 5′-TGATAATGGAGCTCGTT-3′ (SEQ ID NO:61)

The sequencing of cryET59 and cryET60 commenced with the use of primerpr56. The sequencing of cryET83 commenced with the use of primer pr98.Successive oligonucleotides to be used for priming sequencing reactionswere designed from the sequencing data of the previous set of reactions.

The derived sequences were compared to sequences of known cry genesusing the FSTNSCAN program in the PC/GENE sequence analysis package(Intelligenetics, Mountain View, Calif.). This analysis permitted apreliminary classification of the cloned cry genes with respect topreviously-known cry genes (Table 11).

TABLE 6 HOMOLOGY COMPARISON OF DNA SEQUENCES¹ Cloned Gene DNA SequenceIdentity cryET31 90% identity with SEQ ID NO: 4 of WO 98/40490 cryET4099% identity with cry1Aa cryET43 88% identity with cry1Bd1 cryET44 90%identity with cry1Da/1Db cryET45 91% identity with cry1Da/1Db cryET4698% identity with cry1Ga cryET47 99% identity with cry1Ab cryET49 95%identity with cry1Ja cryET51 85% identity with cry1Ac cryET52 84%identity with cry1Da/1Db cryET53 99% identity with SEQ ID NO: 8 of U.S.Pat. No. 5,723,758 cryET54 99.8% identity with cry1Be cryET56 80%identity with cry1Ac cryET57 98% identity with cry1Da cryET59 95%identity with cry9Ca cryET60 99.6% identity with cry9Aa cryET61 97%identity with cry1Ha cryET62 99% identity with cry1Ad cryET63 93%identity with cry1Ac cryET64 91% identity with SEQ ID NO: 9 of U.S. Pat.No. 5,723,758 cryET66 76% identity with cryIGa cryET67 99% identity withSEQ ID NO: 10 of U.S. Pat. No. 5,723,758 cryET72 98% identity with SEQID NO: 4 of WO 98/40490 cryET73 99% identity with SEQ ID NO: 6 of WO98/40490 cryET83 ¹Ktup value set at 2 for FSTNSCAN. The cryET59 andcryET60 sequences were compared using the FASTA program (Ktup = 6) inthe PC/GENE sequence analysis package.

6.9 Example 9

Expression of Cloned Toxin Genes in a B. thuringiensis Host

Plasmid DNA was isolated from E. coli colonies identified byhybridization to a gene-specific probe. The isolated plasmid was thenintroduced into a crystal protein-negative (Cry−) strain of B.thuringiensis using the electroporation protocol of Mettus and Macaluso(1990). Each of the cloning vectors used (see Table 5) has a gene toconfer antibiotic resistance on the cells harboring that plasmid. B.thuringiensis transformants were selected by growth on agar platescontaining 25 mg/ml erythromycin (pHT315) or 5 mg/ml chloramphenicol(pEG597 and pEG1064). Antibiotic-resistant colonies were then evaluatedfor crystal protein production by phase-contrast microscopy. Crystalproducing colonies were then grown in C2 medium (Donovan et al., 1988)to obtain cultures which were analyzed by SDS-PAGE and insect bioassay.

C2 cultures were inoculated with cells from Cry⁺ colonies and grown forthree days at 25-30° C. in the presence of the appropriate antibiotic.During this time the culture grew to stationary phase, sporulated andlysed, releasing the protein inclusions into the medium. The culturesare harvested by centrifugation, which pellets the spores and crystals.The pellets were washed in a solution of 0.005% Triton X-100®, 2 mM EDTAand centrifuged again. The washed pellets were resuspended at one-tenththe original volume in 0.005% Triton X-100®, 2 mM EDTA.

Crystal protein were solubilized from the spores-crystal suspension byincubating the suspension in a solubilization buffer [0.14 M Tris-HCl pH8.0, 2% (wt./vol.) sodium dodecyl sulfate (SDS), 5% (vol./vol.)2-mercaptoethanol, 10% (vol./vol.) glycerol, and 0.1% bromphenol blue]at 100° C. for 5 min. The solubilized crystal proteins weresize-fractionated by SDS-PAGE using a gel with an acrylamideconcentration of 10%. After size fractionation the proteins werevisualized by staining with Coomassie Brilliant Blue R-250.

The expected size for Cry1- and Cry9-related crystal proteins wasapproximately 130 kDa. The expected size for Cry2-related proteins wasapproximately 65 kDa.

6.10 Example 10

Insecticidal Activity of the Cloned B. thuringiensis Toxin Genes

B. thuringiensis recombinant strains producing individual cloned crygenes were grown in C2 medium until the cultures were fully sporulatedand lysed. These C2 cultures were used to evaluate the insecticidalactivity of the crystal proteins produced. Each culture was diluted with0.005% Triton® X-100 to achieve the appropriate dilution for two-dosebioassay screens. Fifty microliters of each dilution were topicallyapplied to 32 wells containing 1.0 ml artificial diet per well (surfacearea of 175 mm²). A single lepidopteran larvae was placed in each of thetreated wells and the tray was covered by a clear perforated mylarsheet. With the exception of the P. xylostella bioassays, that employed3rd instar larvae, all the bioassays were performed with neonate larvae.Larval mortality was scored after 7 days of feeding at 28-30° C. andpercent mortality was expressed as ratio of the number of dead larvae tothe total number of larvae treated (Table 12). In some instances, severestunting of larval growth was observed after 7 days, and the ratio ofstunted/unstunted larva was also recorded. The bioassay results shown inTable 7 demonstrate that the crystal proteins produced by therecombinant B. thuringiensis strains do exhibit insecticidal activityand, furthermore,

TABLE 7 Bioassay evaluations with ET crystal proteins Spodoptera exiguaSpodoptera frugiperda 250 nl/well 2500 nl/well # stunted/ 250 nl/well #stunted/ % mortality % mortality # treated % 2500 nl/wel1 # treatedCry1Ac 0 5 4/32 16 53 1/32 ET31 5 12 17/32  9 6 4/32 ET40 0 5 0 3 3 0ET43 0 8 0 3 3 2/32 ET44 0 2 0 6 0 1/32 ET45 0 0 0 0 0 1/32 ET46 0 12 00 6 0 ET47 19 49 11/32  31 81 6/32 ET49 0 8 0 0 3 0 ET51 0 0 0 0 0 0ET52 0 0 0 3 3 0 ET53 0 0 0 3 0 0 ET54 0 66 3/32 6 34 9/32 ET56 0 0 0 06 0 ET57 2 15 18/32  3 94 0 ET59 0 0 0 0 3 0 ET60 0 0 0 0 3 0 ET61 2 52/32 0 3 0 ET62 2 59 12/32  0 13 0 ET63 0 12 5/32 3 0 0 ET64 0 0 0 3 6 0ET66 0 12 1/32 3 0 1/31 ET67 29 90 0 13 61 0 ET72 0 0 0 3 94 5/31 ET73 02 0 0 0 0 Control 8 8 0 0 0 0 Plutella xylostella Ostrinia nubilalis 250nl/well 2500 nl/well # stunted/ 250 nl/well 2500 nl/well # stunted/ % %mortality # treated % mortality % mortality # treated Cry1Ac 100 100 0100 100 0 ET31 0 2 0 100 100 0 ET40 0 68 0 0 0 2/32 ET43 5 100 0 46 1000 ET44 0 0 0 0 0 3/32 ET45 0 0 0 0 0 4/32 ET46 0 8 0 0 0 0 ET47 100 1000 100 100 0 ET49 0 5 0 0 0 0 ET51 0 0 0 0 0 0 ET52 2 43 0 0 14 16/32ET53 8 97 0 4 46 5/32 ET54 14 100 0 25 89 1/32 ET56 0 0 0 0 0 0 ET57 097 0 0 7 0 ET59 100 100 0 96 100 0 ET60 100 100 0 100 96 0 ET61 0 11 0 00 2/32 ET62 97 100 0 100 100 0 ET63 100 100 0 100 100 0 ET64 40 100 0 68100 0 ET66 100 100 0 86 100 0 ET67 87 100 0 0 79 1/32 ET72 0 0 0 0 0 0ET73 2 2 0 93 100 0 Control 2 2 0 0 0 0 Heliothis virescens Helicoverpazea 250 nl/well 2500 nl/well # stunted/ 250 nl/well 2500 nl/well % %mortality # treated % mortality % mortality Cry1Ac 100 100 0 100 100ET31 97 97 1/32 8 81 ET40 2 5 2/32 2 5 ET43 87 97 1/32 0 2 ET44 8 5 1/325 8 ET45 0 11 0 8 18 ET46 12 25 0 0 8 ET47 87 100 0 83 100 ET49 8 2 0 1115 ET51 2 15 0 5 5 ET52 0 31 1/32 93 11 ET53 22 64 2/32 90 61 ET54 15 645/32 2 5 ET56 0 11 0 8 0 ET57 2 0 0 11 28 ET59 28 84 4/32 2 2 ET60 56 971/32 31 28 ET61 5 5 0 8 5 ET62 44 87 4/32 21 64 ET63 100 100 0 100 100ET64 0 21 0 5 0 ET66 0 8 1/32 0 5 ET67 18 93 1/32 0 68 ET72 34 64 11/32 8 2 ET73 42 90 2/32 8 48 Control 5 5 0 5 5 Agrotis ipsilon Trichoplusiani 250 nl/well 2500 nl/well # stunted/ 250 nl/well 2500 nl/well #stunted/ % % mortality # treated % mortality % mortality # treatedCry1Ac 94 100 100 100 0 ET31 6 6 90 100 0 ET40 0 6 13 32 0 ET43 0 45 100100 0 ET44 6 13 16 26 0 ET45 0 6 13 39 0 ET46 0 0 29 74 0 ET47 0 34 97100 0 ET49 3 0 13 81 0 ET51 0 0 3 19 0 ET52 0 28 81 100 0 ET53 25 81 74100 0 ET54 3 6 100 100 0 ET56 3 3 16 26 0 ET57 13 74 19 100 0 ET59 3 310 84 0 ET60 3 0 97 100 0 ET61 6 28 29 52 0 ET62 23 58 100 100 0 ET63 30 100 100 0 ET64 0 0 87 100 0 ET66 13 91 26 81 0 ET67 3 0 6 100 0 ET72 00 23 74 8/32 ET73 13 6 94 100 0 Control 0 0 3 3 0that the crystal proteins exhibit differential activity towards thelepidopteran species tested.

Additional bioassays were performed with the crystal proteins designatedCryET59, CryET60, CryET66, and CryET83. Crystal proteins produced in C2medium were quantified by SDS-PAGE and densitometry using the methoddescribed by Brussock, S. M. and Currier, T. C., 1990, “Use of SodiumDodecyl Sulfate-Polyacrylamide Gel Electrophoresis to Quantify Bacillusthuringiensis δ-Endotoxins”, in Analytical Chemistry of Bacillusthuringiensis (L. A. Hickle and W. L. Fitch, eds.), The AmericanChemical Society, pp. 78-87.

TABLE 8 Bioassay Evaluation of CryET59 and CryET60 Percent mortality¹Dose Toxin ng/well AI HV HZ ON PX rPX SE TN Control² — 2 6 0 0 2 0 2 0CryET59 100 2 37 0 94 100 100 2 13 CryET59 500 11 80 3 100 100 100 0 63CryET59 5000 62 100 6 100 100 100 71 100 CryET60 500 0 93 22 100 100 1000 100 CryET60 5000 2 100 25 100 100 100 14 100 ¹AI = Agrotis ipsilon, HV= Heliothis virescens, HZ = Helicoverpa zea, ON = Ostrinia nubilalis, PX= Plutella xylostella, rPX = Plutella xylostella colony resistant toCry1A and Cry IF toxins, SE = Spodoptera exigua, TN = Trichoplusia ni.²Control = no toxin added.The procedure was modified to eliminate the neutralization step with 3MHEPES. Crystal proteins resolved by SDS-PAGE were quantified bydensitometry using a Molecular Dynamics model 300A computingdensitometer and purified bovine serum albumin (Pierce, Rockford, Ill.)as a standard.

The bioassay results shown in Table 8 demonstrate that CryET59 andCryET60 are toxic to a number of lepidopteran species, including acolony of P. xylostella that is resistant to Cry1A and Cry1F crystalproteins. Eight-dose assays with CryET66 also demonstrated excellenttoxicity towards both the susceptible and resistant colonies of P.xylostella (Table 14). In this instance, eight crystal proteinconcentrations were prepared by serial dilution of the crystal proteinsuspensions in 0.005% Triton® X-100 and 50 ul of each concentration wastopically applied to wells containing 1.0 ml of artificial diet. Afterthe wells had dried, a single larvae was placed in each of the treatedwells and the tray was covered by a clear perforated mylar sheet (32larvae for each crystal protein concentration). Larval mortality wasscored after 7 days of feeding at 28-30° C. Mortality data was expressedas LC₅₀ and LC₉₅ values, the concentration of crystal protein (ng/175mm² diet well) causing 50% and 95% mortality, respectively (Daum, 1970).

TABLE 9 Toxin LC₅₀ ¹ 95% C.I. LC₉₅ ² Slope Toxicity of CryET66 towardsPlutella xylostella Cry1Ac 8.05  5.0-15.2 52.94 2.01 Cry1C 25.0615.7-40.6 117.07 2.46 CryET66 0.42 0.4-0.5 1.4 3.13 Toxicity of CryET66towards Cry1A-resistant Plutella xylostella Cry1Ac *No significantmortality Cry1C 27.32 15.4-51.1 156.13 2.17 CryET66 1.65 1.3-2.0 6.412.79 ¹the concentration of crystal protein, in nanograms of crystalprotein per well, required to achieve 50% mortality ²the concentrationof crystal protein, in nanograms of crystal protein per well, requiredto achieve 95% mortality.Table 15 shows that the CryET83 protein exhibits toxicity towards a widevariety of lepidopteran pests and may exhibit improved toxicity towardsS. exigua and H. virescens when compared to the other Cry9-type proteinsCryET59 and CryET60.

TABLE 10 Toxicity of CryET83 towards lepidopteran larvae¹ Dose² AI³ HVHZ ON PX SE SF TN 5 5 10 9 50 53 75 69 100 91 500 0 100 67 100 5000 32100 10000 84 100 ¹Toxicity calculated as percent mortality among treatedlarvae. ²ng CryET83 crystal protein/175 mm² diet well ³Abbreviationsdescribed in Table 8; SF = Spodoptera frugiperdaThe recombinant B. thuringiensis strains listed in Table 5 weredeposited with the ARS Patent Culture Collection and had been assignedthe NRRL deposit numbers shown in Table 11.

TABLE 11 Biological Deposits Polypeptide Polypeptide PolynucleotideRecomb. NRRL Deposit Designation Seq. ID No.: Seq ID No.: Strain No.:Cry ET31 2 1 EG11562 B-21921 Cry ET40 4 3 EG11901 B-21922 Cry ET43 6 5EG7692 B-21923 Cry ET44 8 7 EG11629 B-21924 Cry ET45 10 9 EG7694 B-21925Cry ET46 12 11 EG7695 B-21926 Cry ET47 14 13 EG7696 B-21927 Cry ET49 1615 EG11630 B-21928 Cry ET51 18 17 EG11921 B-21929 Cry ET52 20 19 EG11584B-21930 Cry ET53 22 21 EG11906 B-21931 Cry ET54 63 62 EG11907 B-21932Cry ET56 24 23 EG11909 B-21933 Cry ET57 26 25 EG11910 B-21934 Cry ET5928 27 EG12102 B-21935 Cry ET60 30 29 EG12103 B-21936 Cry ET61 32 31EG11634 B-21937 Cry ET62 34 33 EG11635 B-21938 Cry ET63 36 35 EG11636B-21939 Cry ET64 38 37 EG11638 B-21940 Cry ET66 40 39 EG11640 B-21941Cry ET67 42 41 EG11642 B-21942 Cry ET68 44 43 EG11644 B-30137 Cry ET7246 45 EG11440 B-21943 Cry ET73 48 47 EG11465 B-21944 Cry ET83 50 49EG11785 B-30138

6.11 Example 11

Modification of Cry Genes for Expression in Plants

Wild-type cry genes are known to be expressed poorly in plants as a fulllength gene or as a truncated gene. Typically, the G+C content of a crygene is low (37%) and often contains many A+T rich regions, potentialpolyadenylation sites and numerous ATTTA sequences. Table 12 shows alist of potential polyadenylation sequences which should be avoided whenpreparing the “plantized” gene construct.

TABLE 12 List of Sequences of Potential Polyadenylation Signals AATAAA*AAGCAT AATAAT* ATTAAT AACCAA ATACAT ATATAA AAAATA AATCAA ATTAAA** ATACTAAATTAA** ATAAAA AATACA** ATGAAA CATAAA** * indicates a potential majorplant polyadenylation site. ** indicates a potential minor animalpolyadenylation site. All others are potential minor plantpolyadenylation sites.

The regions for mutagenesis may be selected in the following manner. Allregions of the DNA sequence of the cry gene are identified whichcontained five or more consecutive base pairs which were A or T. Thesewere ranked in terms of length and highest percentage of A+T in thesurrounding sequence over a 20-30 base pair region. The DNA is analysedfor regions which might contain polyadenylation sites or ATTTAsequences. Oligonucleotides are then designed which maximize theelimination of A+T consecutive regions which contained one or morepolyadenylation sites or ATTTA sequences. Two potential plantpolyadenylation sites have been shown to be more critical based onpublished reports. Codons are selected which increase G+C content, butdo not generate restriction sites for enzymes useful for cloning andassembly of the modified gene (e.g., BamHI, BglII, SacI, NcoI, EcoRV,etc.). Likewise condons are avoided which contain the doublets TA or GCwhich have been reported to be infrequently-found codons in plants.

Although the CaMV35S promoter is generally a high level constitutivepromoter in most plant tissues, the expression level of genes driven theCaMV35S promoter is low in floral tissue relative to the levels seen inleaf tissue. Because the economically important targets damaged by someinsects are the floral parts or derived from floral parts (e.g., cottonsquares and bolls, tobacco buds, tomato buds and fruit), it is oftenadvantageous to increase the expression of crystal proteins in thesetissues over that obtained with the CaMV35S promoter.

The 35S promoter of Figwort Mosaic Virus (FMV) is analogous to theCaMV35S promoter. This promoter has been isolated and engineered into aplant transformation vector. Relative to the CaMV promoter, the FMV 35Spromoter is highly expressed in the floral tissue, while still providingsimilar high levels of gene expression in other tissues such as leaf. Aplant transformation vector, may be constructed in which the full lengthsynthetic cry gene is driven by the FMV 35S promoter. Tobacco plants maybe transformed with the vector and compared for expression of thecrystal protein by Western blot or ELISA immunoassay in leaf and floraltissue. The FMV promoter has been used to produce relatively high levelsof crystal protein in floral tissue compared to the CaMV promoter.

6.12 Example 12

Expression of Synthetic Cry Genes with ssRUBISCO Promoters andChloroplast Transit Peptides

The genes in plants encoding the small subunit of RUBISCO(SSU) are oftenhighly expressed, light regulated and sometimes show tissue specificity.These expression properties are largely due to the promoter sequences ofthese genes. It has been possible to use SSU promoters to expressheterologous genes in transformed plants. Typically a plant will containmultiple SSU genes, and the expression levels and tissue specificity ofdifferent SSU genes will be different. The SSU proteins are encoded inthe nucleus and synthesized in the cytoplasm as precursors that containan N-terminal extension known as the chloroplast transit peptide (CTP).The CTP directs the precursor to the chloroplast and promotes the uptakeof the SSU protein into the chloroplast. In this process, the CTP iscleaved from the SSU protein. These CTP sequences have been used todirect heterologous proteins into chloroplasts of transformed plants.

The SSU promoters might have several advantages for expression ofheterologous genes in plants. Some SSU promoters are very highlyexpressed and could give rise to expression levels as high or higherthan those observed with the CaMV35S promoter. The tissue distributionof expression from SSU promoters is different from that of the CaMV35Spromoter, so for control of some insect pests, it may be advantageous todirect the expression of crystal proteins to those cells in which SSU ismost highly expressed. For example, although relatively constitutive, inthe leaf the CaMV35S promoter is more highly expressed in vasculartissue than in some other parts of the leaf, while most SSU promotersare most highly expressed in the mesophyll cells of the leaf. Some SSUpromoters also are more highly tissue specific, so it could be possibleto utilize a specific SSU promoter to express the protein of the presentinvention in only a subset of plant tissues, if for example expressionof such a protein in certain cells was found to be deleterious to thosecells. For example, for control of Colorado potato beetle in potato, itmay be advantageous to use SSU promoters to direct crystal proteinexpression to the leaves but not to the edible tubers.

Utilizing SSU CTP sequences to localize crystal proteins to thechloroplast might also be advantageous. Localization of the B.thuringiensis crystal proteins to the chloroplast could protect thesefrom proteases found in the cytoplasm. This could stabilize the proteinsand lead to higher levels of accumulation of active toxin. cry genescontaining the CTP may be used in combination with the SSU promoter orwith other promoters such as CaMV35S.

6.13 Example 13

Targeting of Cry Proteins to the Extracellular Space or Vacuole Throughthe Use of Signal Peptides

The B. thuringiensis proteins produced from the synthetic genesdescribed here are localized to the cytoplasm of the plant cell, andthis cytoplasmic localization results in plants that are insecticidallyeffective. It may be advantageous for some purposes to direct the B.thuringiensis proteins to other compartments of the plant cell.Localizing B. thuringiensis proteins in compartments other than thecytoplasm may result in less exposure of the B. thuringiensis proteinsto cytoplasmic proteases leading to greater accumulation of the proteinyielding enhanced insecticidal activity. Extracellular localizationcould lead to more efficient exposure of certain insects to the B.thuringiensis proteins leading to greater efficacy. If a B.thuringiensis protein were found to be deleterious to plant cellfunction, then localization to a noncytoplasmic compartment couldprotect these cells from the protein.

In plants as well as other eukaryotes, proteins that are destined to belocalized either extracellularly or in several specific compartments aretypically synthesized with an N-terminal amino acid extension known asthe signal peptide. This signal peptide directs the protein to enter thecompartmentalization pathway, and it is typically cleaved from themature protein as an early step in compartmentalization. For anextracellular protein, the secretory pathway typically involvescotranslational insertion into the endoplasmic reticulum with cleavageof the signal peptide occurring at this stage. The mature protein thenpasses through the Golgi body into vesicles that fuse with the plasmamembrane thus releasing the protein into the extracellular space.Proteins destined for other compartments follow a similar pathway. Forexample, proteins that are destined for the endoplasmic reticulum or theGolgi body follow this scheme, but they are specifically retained in theappropriate compartment. In plants, some proteins are also targeted tothe vacuole, another membrane bound compartment in the cytoplasm of manyplant cells. Vacuole targeted proteins diverge from the above pathway atthe Golgi body where they enter vesicles that fuse with the vacuole.

A common feature of this protein targeting is the signal peptide thatinitiates the compartmentalization process. Fusing a signal peptide to aprotein will in many cases lead to the targeting of that protein to theendoplasmic reticulum. The efficiency of this step may depend on thesequence of the mature protein itself as well. The signals that direct aprotein to a specific compartment rather than to the extracellular spaceare not as clearly defined. It appears that many of the signals thatdirect the protein to specific compartments are contained within theamino acid sequence of the mature protein. This has been shown for somevacuole targeted proteins, but it is not yet possible to define thesesequences precisely. It appears that secretion into the extracellularspace is the “default” pathway for a protein that contains a signalsequence but no other compartmentalization signals. Thus, a strategy todirect B. thuringiensis proteins out of the cytoplasm is to fuse thegenes for synthetic B. thuringiensis genes to DNA sequences encodingknown plant signal peptides. These fusion genes will give rise to B.thuringiensis proteins that enter the secretory pathway, and lead toextracellular secretion or targeting to the vacuole or othercompartments.

Signal sequences for several plant genes have been described. One suchsequence is for the tobacco pathogenesis related protein PR1b has beenpreviously described (Cornelissen et al., 1986). The PR1b protein isnormally localized to the extracellular space. Another type of signalpeptide is contained on seed storage proteins of legumes. These proteinsare localized to the protein body of seeds, which is a vacuole likecompartment found in seeds. A signal peptide DNA sequence for theβ-subunit of the 7S storage protein of common bean (Phaseolus vulgaris),PvuB has been described (Doyle et al., 1986). Based on the publishedthese published sequences, genes may be synthesized chemically usingoligonucleotides that encode the signal peptides for PR1b and PvuB. Insome cases to achieve secretion or compartmentalization of heterologousproteins, it may be necessary to include some amino acid sequence beyondthe normal cleavage site of the signal peptide. This may be necessary toinsure proper cleavage of the signal peptide.

6.14 Example 14

Isolation of Transgenic Plants Resistant to Insects Using Crytransgenes

6.64.1 Plant Gene Construction

The expression of a plant gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3′non-translated region which adds polyadenylate nucleotides to the 3′ endof the RNA. Transcription of DNA into mRNA is regulated by a region ofDNA usually referred to as the “promoter”. The promoter region containsa sequence of bases that signals RNA polymerase to associate with theDNA and to initiate the transcription of mRNA using one of the DNAstrands as a template to make a corresponding strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. Such promoters may be obtained from plantsor plant viruses and include, but are not limited to, the nopalinesynthase (NOS) and octopine synthase (OCS) promoters (which are carriedon tumor-inducing plasmids of Agrobacterium tumefaciens), thecauliflower mosaic virus (CaMV) 19S and 35S promoters, thelight-inducible promoter from the small subunit of ribulose1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant plantpolypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All ofthese promoters have been used to create various types of DNA constructswhich have been expressed in plants (see e.g., U.S. Pat. No. 5,463,175,specifically incorporated herein by reference).

The particular promoter selected should be capable of causing sufficientexpression of the enzyme coding sequence to result in the production ofan effective amount of protein. One set of preferred promoters areconstitutive promoters such as the CaMV35S or FMV35S promoters thatyield high levels of expression in most plant organs (U.S. Pat. No.5,378,619, specifically incorporated herein by reference). Another setof preferred promoters are root enhanced or specific promoters such asthe CaMV derived 4 as-1 promoter or the wheat POX1 promoter (U.S. Pat.No. 5,023,179, specifically incorporated herein by reference; Hertig etal., 1991). The root enhanced or specific promoters would beparticularly preferred for the control of corn rootworm (Diabroticusspp.) in transgenic corn plants.

The promoters used in the DNA constructs (i.e. chimeric plant genes) ofthe present invention may be modified, if desired, to affect theircontrol characteristics. For example, the CaMV35S promoter may beligated to the portion of the ssRUBISCO gene that represses theexpression of ssRUBISCO in the absence of light, to create a promoterwhich is active in leaves but not in roots. The resulting chimericpromoter may be used as described herein. For purposes of thisdescription, the phrase “CaMV35S” promoter thus includes variations ofCaMV35S promoter, e.g., promoters derived by means of ligation withoperator regions, random or controlled mutagenesis, etc. Furthermore,the promoters may be altered to contain multiple “enhancer sequences” toassist in elevating gene expression.

The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNA's, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence.

For optimized expression in monocotyledenous plants such as maize, anintron should also be included in the DNA expression construct. Thisintron would typically be placed near the 5′ end of the mRNA inuntranslated sequence. This intron could be obtained from, but notlimited to, a set of introns consisting of the maize hsp70 intron (U.S.Pat. No. 5,424,412; specifically incorporated herein by reference) orthe rice Act1 intron (McElroy et al., 1990). As shown below, the maizehsp70 intron is useful in the present invention.

As noted above, the 3′ non-translated region of the chimeric plant genesof the present invention contains a polyadenylation signal whichfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. Examples of preferred 3′ regions are (1) the 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene and (2) plant genes such as the pea ssRUBISCO E9gene (Fischhoff et al., 1987).

6.14.2 Plant Transformation and Expression

A plant gene containing a structural coding sequence of the presentinvention can be inserted into the genome of a plant by any suitablemethod. Suitable plant transformation vectors include those derived froma Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed,e.g., by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and Eur.Pat. Appl. Publ. No. EP0120516. In addition to plant transformationvectors derived from the Ti or root-inducing (Ri) plasmids ofAgrobacterium, alternative methods can be used to insert the DNAconstructs of this invention into plant cells. Such methods may involve,for example, the use of liposomes, electroporation, chemicals thatincrease free DNA uptake, free DNA delivery via microprojectilebombardment, and transformation using viruses or pollen (Fromm et al.,1986; Armstrong et al., 1990; Fromm et al., 1990).

6.14.3 Construction of Monocot Plant Expression Vectors for Cry Genes

For efficient expression of cry genes in transgenic plants, the genemust have a suitable sequence composition (Diehn et al., 1996). To placethe cry gene in a vector suitable for expression in monocotyledonousplants (i.e. under control of the enhanced Cauliflower Mosaic Virus 35Spromoter and link to the hsp70 intron followed by a nopaline synthasepolyadenylation site as in U.S. Pat. No. 5,424,412, specificallyincorporated herein by reference), a vector such as pMON19469 may beused. Such a vector is conveniently digested with NcoI and EcoRIrestriction enzymes. The larger vector band of approximately 4.6 kb isthen electrophoresed, purified, and ligated with T4 DNA ligase to anNcoI-EcoRI fragment which contains the synthetic cry gene. The ligationmix is then transformed into E. coli, carbenicillin resistant coloniesrecovered and plasmid DNA recovered by DNA miniprep procedures. The DNAis then subjected to restriction endonuclease analysis with enzymes suchas NcoI and EcoRI (together), NotI, and/or PstI individually or incombination, to identify clones containing the cry coding sequence fusedto an intron such as the hsp70 intron, placed under the control of theenhanced CaMV35S promoter.

To place the gene in a vector suitable for recovery of stablytransformed and insect resistant plants, the 3.75-kb NotI restrictionfragment from pMON33708 containing the lysine oxidase coding sequencefused to the hsp70 intron under control of the enhanced CaMV35S promotermay be isolated by gel electrophoresis and purification. This fragmentis then ligated with a vector such as pMON30460 which has beenpreviously treated with NotI and calf intestinal alkaline phosphatase(pMON30460 contains the neomycin phosphotransferase coding sequenceunder control of the CaMV35S promoter). Kanamycin resistant colonies maythen be obtained by transformation of this ligation mix into E. coli andcolonies containing the desired plasmid may be identified by restrictionendonuclease digestion of plasmid miniprep DNAs. Restriction enzymessuch as NotI, EcoRV, HindIII, NcoI, EcoRI, and BglII may be used toidentify the appropriate clones in which the orientation of both genesare in tandem (i.e. the 3′ end of the cry expression cassette is linkedto the 5′ end of the nptII expression cassette). Expression of the Cryprotein by the resulting plasmid in corn protoplasts may be confirmed byelectroporation of the vector DNA into protoplasts followed by proteinblot and ELISA analysis. This vector may be introduced into the genomicDNA of corn embryos by particle gun bombardment followed by paromomycinselection to obtain corn plants expressing the cry gene essentially asdescribed in U.S. Pat. No. 5,424,412, specifically incorporated hereinby reference.

As an example, the vector may be introduced via cobombardment with ahygromycin resistance conferring plasmid into immature embryo scutella(IES) of maize, followed by hygromycin selection, and regeneration.Transgenic corn lines expressing the cry protein may then be identifiedby ELISA analysis. Progeny seed from these events may then besubsequently tested for protection from insect feeding.

7.0 REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims. Accordingly, the exclusive rights sought to be patentedare as described in the claims below.

1-33. (canceled)
 34. A transgenic plant having incorporated into itsgenome a polynucleotide encoding the polypeptide of SEQ ID NO:2 or aninsecticidal fragment thereof operably linked to a plant-expressiblepromoter. 35-37. (canceled)
 38. The transgenic plant of claim 34,further defined as a monocotyledonous plant.
 39. The transgenic plant ofclaim 38, further defined as a corn, wheat, oat, rice, barley, turfgrass, or pasture grass plant.
 40. The transgenic plant of claim 34,further defined as a dicotyledonous plant.
 41. The transgenic plant ofclaim 40, further defined as a legume, soybean, tobacco, tomato, potato,cotton, fruit, berry, vegetable or tree plant.
 42. A progeny of anygeneration of the transgenic plant of claim 34, wherein said progenycomprises said polynucleotide.
 43. A seed of any generation of thetransgenic plant of claim 34, wherein said seed comprises saidpolynucleotide. 44-51. (canceled)
 52. A method of preparing an insectresistant plant, said method comprising: (a) transforming recipientplant cells with a polynucleotide encoding the polypeptide of SEQ IDNO:2 or an insecticidal fragment thereof operably linked to aplant-expressible promoter; (b) selecting a recipient plant cellcomprising said polynucleotide; and (c) regenerating a plant from theselected recipient plant cell; wherein said plant comprises saidpolynucleotide and is insect resistant.
 53. (canceled)
 54. Thetransgenic plant of claim 34, wherein said polynucleotide is furtheroperably linked to a polynucleotide encoding a signal peptide.
 55. Thetransgenic plant of claim 54, wherein said signal peptide targetsexpression of said polypeptide or said insecticidal fragment to thechloroplast, endoplasmic reticulum, Golgi body or vacuole, or forsecretion.
 56. The transgenic plant of claim 55, wherein said signalpeptide is a chloroplast transit peptide or a secretory signal peptide.57. (canceled)
 58. The method of claim 52, wherein said polynucleotideis further operably linked to a polynucleotide encoding a signalpeptide.
 59. The method of claim 58, wherein said signal peptide is achloroplast transit peptide or a secretory signal peptide.
 60. A methodfor controlling Lepidopteran insect infestation in a field of cropplants, said method comprising expressing in said plants aninsecticidally effective amount of the polypeptide of SEQ ID NO:2 or aninsecticidal fragment thereof.
 61. The method of claim 60, wherein saidplants are transformed with a polynucleotide encoding said polypeptideor said insecticidal fragment operably linked to a plant-expressiblepromoter.
 62. (canceled)
 63. The method of claim 61, wherein saidpolynucleotide is further operably linked to a polynucleotide encoding asignal peptide.
 64. The method of claim 63, wherein said signal peptideis a chloroplast transit peptide or a secretory signal peptide.
 65. Thetransgenic plant of claim 34, wherein said plant-expressible promoter isselected from the group consisting of corn sucrose synthetase 1, cornalcohol dehydrogenase 1, corn light harvesting complex, corn heat shockprotein, pea small subunit RuBP carboxylase, Ti plasmid mannopinesynthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, beanglycine rich protein 1, Potato patatin, lectin, CaMV 35S, and the S-E9small subunit RuBP carboxylase promoter.
 66. The method of claim 52,wherein said plant-expressible promoter is selected from the groupconsisting of corn sucrose synthetase 1, corn alcohol dehydrogenase 1,corn light harvesting complex, corn heat shock protein, pea smallsubunit RuBP carboxylase, Ti plasmid mannopine synthase, Ti plasmidnopaline synthase, petunia chalcone isomerase, bean glycine rich protein1, Potato patatin, lectin, CaMV 35S, and the S-E9 small subunit RuBPcarboxylase promoter.
 67. The method of claim 61, wherein saidplant-expressible promoter is selected from the group consisting of cornsucrose synthetase 1, corn alcohol dehydrogenase 1, corn lightharvesting complex, corn heat shock protein, pea small subunit RuBPcarboxylase, Ti plasmid mannopine synthase, Ti plasmid nopalinesynthase, petunia chalcone isomerase, bean glycine rich protein 1,Potato patatin, lectin, CaMV 35S, and the S-E9 small subunit RuBPcarboxylase promoter.